Chromobacterium subtsugage genes

ABSTRACT

Disclosed herein are the nucleotide sequences of the  Chromobacterium subtsugae  genes. In addition, the amino acid sequences of proteins encoded by the  C. subtsugae  genes are provided. Nucleic acids, vectors and polypeptides comprising the aforementioned sequences are also provided. Homologues, functional fragments and conservative variants of the aforementioned sequences are also provided. Compositions having pesticidal, bioremedial and plant growth-promoting activities comprising  C. subtsugae  genes and proteins, and methods for the use of these compositions, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2015/047649, filed on Aug. 31, 2015 which claims the benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/046,672, filedSep. 5, 2014. All of which are hereby incorporated by reference in theirentirety.

TECHNICAL FIELD OF THE INVENTION

The present disclosure is in the field of biopesticides; in particularbacterial pesticides, their genes, gene products, and method of usethereof.

REFERENCE TO A SEQUENCE LISTING

This instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy is namedMBI-203-0005-PCT_seq_ST25.txt and is 335 kilobytes in size.

BACKGROUND OF THE INVENTION

Chromobacterium subtsugae

In 2000, a purple-pigmented bacterium (PRAA4-1) was isolated from forestsoil in Maryland (Martin et al., 2004). In initial screens, thisbacterium was found to be toxic to Colorado potato beetle and otherinsect pests (Martin et al., 2007a). Additional work with the isolaterevealed activity gainst mites, grubs, diverse beetle species, aphidsand plant parasitic nematodes, among other plant pests (Martin et al.,2007b, US Patent Application Publication No. 2012/0100236 A1). SomePRAA4-1 protein studies exist in the field of art with respect toprotein actives against insects.

Proteases and Insect Control

Proteases have the ability to target and destroy essential proteins andtissues of insects. Plants have naturally evolved to express proteasesto protect against insects. Insect predators also produce protease intheir venom, which contributes to mortality. Proteases have beenidentified as important insecticidal agents for control of insects inagriculture.

Proteases with insecticidal activity fall into three general categories:cysteine proteases, metalloproteases and serine proteases. Proteases ofthese classes target the midgut, cuticle and hemocoel. The peritrophicmatrix of the midgut is an ideal target for insect control because itlines and protects the midgut epithelium from food particles, digestiveenzymes and pathogens; in addition to acting as a biochemical barrier(Hegedus at al., 2009). Enhancins are zinc metalloproteases expressed bybaculoviruses that facilitate nucleopolyhedrovirus infections inlepidopterans (Lepore et al., 1996). These proteases promote theinfection of lepidopteran larvae by digesting the invertebrateintestinal mucin protein of the peritrophic matrix, which in turnpromotes infection of the midgut epithelium (Wang and Granados, 1997).Homologs of enhancin genes found in baculovirus have been identified inthe genomes of Yersinia pestis, Bacillus anthracis, Bacillusthuringiensis and Bacillus cereus (Galloway et al., 2005; Hajaij-Ellouzeet al., 2006).

Plant cysteine proteases also demonstrate activity against lepidopteranlarvae. Cysteine proteases in the latex of the papaya and wild fig treesare essential in the defense against various lepidopteran larvae.Toxicity to the larvae was lost when the latex was washed or when theleaves were treated with a cysteine protease-inhibitor, indicating thatthe defense may be due to the high concentration of cysteine proteasesin the latex (Konno et al., 2004).

Proteases that target the cuticle are also important in insect control.The cuticle covers the entire outside of the insect as well as someinvaginations of internal structures. The cuticle is composed of a waxyepicuticle, an exocuticle and an endocuticle that consist of protein,lipid and chitin (Harrison and Bonning 2010). Fungal infection ofinsects by Metarhizium anisopliae and Beauveria bassiana occurs when thefungal spores germinate on the cuticle, forming structures forpenetration of the cuticle by a variety of enzymes, including proteases(Freimoser at al., 2003; Cho et al., 2006). One notable serine proteaseproduced by M. anisopliae, PR1A, digests the cuticle and plays anessential role in penetration (St. Leger et al. 1987). A clone of M.anisopliae was engineered to contain additional copies of the pyla geneand showed 25% more kill of tobacco hornworm than the wild-type (StLeger et al., 1996). B. basianna was also engineered to express the M.anisopliae PR1A protease and demonstrated increased toxicity of larvaeof the Masson's, pine caterpillar, Dendrolimus punctatus, and the waxmoth, Galleria mellonella (Lu et al., 2008).

The basement membrane of insects consists of proteins that surround thetissue and contribute to a variety of functions from structural supportto barriers for viruses. Three potential basement membrane-degradingproteins were evaluated using Autographa californica multiplenucleopolyhedrovirus (AcMNPV). This baculovirus was engineered toexpress two vertebrate metalloproteases, rat stromelysin and humangeatinase A, as well as the fruit fly cathepsin L, ScathL. The ScathLprotease demonstrated the best baculovirus activity. The median survivaltime of infected tobacco budworm larvae was reduced by 50% when comparedto wild-type infected larvae (Harrison and Bonning, 2001). This datasupports the idea that proteases expressed in viruses have the abilityto access the basement membrane of insects, which generally functions asa barrier to viruses. A previous report identified two basement membraneproteins of imaginal discs of fruit fly larvae that are susceptible tohydrolysis by cathepsin L (Homma and Natori, 1996). Purified ScathLprotease was also toxic to a variety of insect pests when it wasinjected into the hemocoel. The purified protease demonstrated similarmelanization, mortality and hemolymph protease activity in lepidopteranlarvae as was seen ScathL expressed baculovirus infections (Li et al.,2008). Basement membrane damage is cause by purified ScathL proteaseboth in vivo and in vitro (Tang et al., 2007; Philip et al. 2007).

Arthropod predators have also been shown to contain basement membranecleaving proteases in their venom. One example is the parasitic wasp,Eulophus pennicornis, in which 3 metalloproteinases (EpMP1-3) wereidentified in the venom glands. Recombinant EpMP3 was injected into thehemocoel of Lacanobia oleracea larvae and resulted in significantmortality, or impaired development and growth in surviving larvae (Priceet al., 2009). Social aphid soldier nymphs produce a toxic cathepsin Bprotease (cysteine protease) in their intestines. The protease is orallyexcreted into enemies and demonstrates insecticidal activity (Kutsukakeet al., 2008).

A protease isolated from the bacterium, Xenorhabdus nematophilia, hasbeen shown to suppress antibacterial peptides involved in insect immuneresponse, making the insect susceptible to the pathogenetic process(Caldas et al., 2002). The enterobacterium, Photorhabdus luminscense,has been shown to be pathogenic to a broad spectrum of insects. Thegenome sequence of this bacterium identified genes related to toxicity,including proteases (Duchaud et al., 2003).

The use of proteases as insecticides has been of interest to plantmodifications as well. Basement-membrane degrading proteases have beencharacterized and engineered for transgenic insecticidal protocols, withthe goal of developing transgenic plants that are resistant to insectpests (U.S. Pat. No. 6,673,340, Harrison and Bonning, 2004). Proteasesin the gut of insects have been shown to affect the impact of Bacillusthuringiensis Cry insecticidal proteins. Some proteases activate Cryproteins by processing them from a protoxin to a toxic form. Insecttoxins have been modified to comprise proteolytic activation sites withthe goal of incorporating this modification into transformed plants,plant cells and seeds. Cleavage of these sites by the insect gutprotease results in an active insect toxin within the gut of the pest(U.S. Pat. No. 7,473,821, Abad et al., 2009).

Insecticidal Activity of Chitinases

Chitinases expedite insecticidal activity by puncturing the insectmidgut lining and degrading the insect cuticle. Degradation of thesemembranes exposes the insects to pathogens, to other insecticidalcompounds, and/or to plant defenses.

Chitinases hydrolyze the structural polysaccharide chitin, a linearhomopolymer of 2-acetamido-2-deoxy-D-glucopyranoside, linked byβ-1→4-linkages, which is a component of the exoskeleton and gut liningof insects. Chitinases are classified as either family 18 or family 19glycosyl hydrolases. Family 18 chitinases are widespread, found inbacteria, plants, and animals; while family 19 chitinases are mainlyfound in plants (Henrissat and Bairoch, 1993). In insects, Chitinasesplay a role in molting (Samuels and Reynolds, 1993, Merzendorfer andZimoch, 2003).

Chitinases alone show some insecticidal activity. Chitinase fromSerretia marcenscens was found to be toxic to seventh instar Galleriamellonella larvae (Lysenk, 1976). Transgenic plants which express insectchitinases have been shown to have increased resistance to insectpestss. Tobacco plants were transformed with cDNA encoding a Manducasexta chitinase. Leaves from these transgenic plants were infested withHeliothis virescens larvae. After 3 weeks it was found that chitinasepositive leaves had less larval biomass and feeding damage thanchitinase negative leaves. It is possible that the activity of thechitinases render insects more susceptible to plant defenses (Ding, etal., 1997).

Insect cuticles provide a physical barrier to protect the insect formpathogens or other environmental hazards, and are composed primarily ofchitin (Kramer, et al., 1995). Entomopathogenic fungi Metarhiziumanisopliae, Beauvaria bassiana, Beauvaria amorpha, Verticillium lecanii,and Aspergillus flavus all secrete chitinases to break down the cuticleand enter the insect host (St Leger, et al., 1986, 1992, Campos, et al.2005). According to Kim, et al., chitinase-containing supernatants ofBeauveria bassina were toxic to Aphis gossypii adults. However, whenthese supernatants were treated with an excess of chitin to inhibit theactivity of the fungal chitinases, this mortality was significantlyreduced, suggesting that chitinase plays an integral role in breakingdown the cuticle and facilitating infection (Kim, et al. 2010).Chitinases have also been isolated from the venom of the endoparasiticwasp Chelonus sp., where they possibly help the venom penetrate thedefenses of chitin protected prey (Krishnan, et al., 1994).

The peritrophic membrane, which lines the insect midgut, is anotherprimarily-chitin-composed barrier that protects insects from pathogens.Any enzyme that can puncture this membrane has potential as abioinsecticide (Wang and Granados, 2001). Hubner, et al. demonstratedthat malarial parasites excrete chitinases to penetrate the peritrophicmembrane in mosquitoes (Hubner, et al., 1991), and Shahabuddin, et al.confirmed that inhibition of chitinase with allosamidin is sufficient toprevent the malarial parasite Plasmodium gallinaceum from crossing theperitrophic membrane of Anopheles freeborni. Also, the addition ofexogenous chitinase from Streptomyces griseus during the development ofthe Anopheles freeborni midgut prevented the formation of theperitrophic membrane (Shahabuddin, et al., 1993). This demonstrates thatchitinases can break down the peritrophic membrane. Regev, et al. usedE. coli to express Serratia marcescens endochitinase ChiA and confirmedwith electron microscopy that Spodoptera littoralis larvae exposed tothe endochitinase exhibited perforations in the peritrophic membrane(Regev, et al., 1996).

Because of the ability of chitinase to perforate the peritrophicmembrane, endochitinases have also been shown to increase theinsecticidal activity of Bacillus thuringiensis (Bt). Choristoneurafumiferana larvae reared on Agies balsamea treated with a mixture of adiluted commercial formulation of Bt and chitinase were killed morequickly than larvae reared on foliage treated with just Bt alone(Smirnoff, 1973). A mixture of a low concentration of Bt and S.marcenscens chitinase also resulted in higher mortality of Spodopteralittoralis larvae than Bt alone (Sheh et al., 1983). It is believed thatthis synergistic effect is due to puncturing of the peritrophic liningof the insect gut by the chitinase, facilitating the penetration of Btspores into the insect. (Smirnoff, 1973).

Yen-Tc, an ABC type protein that is both necessary and sufficient forthe entomopathogenicity of Yersinia entomophaga in the insect Costelytrazealandica, contains two family 18 chitinases, making it the firstinsecticidal toxin complex identified to incorporate chitinases. It ishypothesized that the chitinases are responsible for breaking downperitrophic membrane and exposing the midgut epithelial cells to thetoxin. However, the chitinases may only be active in regions of themidgut with a relatively neutral pH (Busby, 2012).

Chitinases are also integral to the activity of some insect viruses.Hatwin, et al. created mutants of the Autographa californicanucleopolyhedrovirus (AcMNPV) that lacked the gene for chitinase.Usually, this virus causes liquefaction of the host larvae, facilitatingthe spread of the virus. This liquefaction did not occur whenTrichoplusia ni larvae were infected with the chitinase negative virus.It was also confirmed that the AcMNPV chitinase is active under thealkaline conditions of the insect midgut (Hatwin, et al. 1997). Arecombinant version of the same Autographa californicanucleopolyhedrovirus that expressed a Haemaphysalis longicornischitinase was found to have bioarcaricidal activity againstHaemaphysalis longicornis nymphs (Assegna, et al. 2006).

Rhs-Like Genes Encode Insecticidal Toxins

The rhs (rearrangement hotspot) gene family was first identified in E.coli. These genes confer chromosomal rearrangements by homologousexchange (Lin et al., 1984). They are 2 to 12 kb in size and exhibit along core with a short tip. The core sequences are GC rich and highlyconserved, but the tip sequences are GC-poor and highly variable. Theyencode proteins that have a large core domain and a short C-terminal tipdomain. The protein core domain is hydrophilic and contains YD-repeats(Jackson et al., 2009). The Rhs proteins are capable of interacting withbacterial cell surfaces and binding to specific ligands (Wang et al.,1998). While the function of the Rhs proteins remains unknown (Hill etal., 1994), the structure is important because the YD repeats and highlyconserved sequences resemble rhs and rhs-like genes encodinginsecticidal toxins produced by bacteria.

Photorhabdus luminescens is a mutualistic symbiont of the nematodes fromthe Heterorhabditae family. The nematode infects the insect and injectsthe bacterium into the hemocoel of the insect. The bacterium thensecretes toxins that kill the insect (Frost et al., 1997). Bowen et al.(1998), purified a high molecular weight protein associated with oraland injectable insecticidal toxicity that targets insects. In anotherstudy, Bowen et al. (1998) used high performance liquid chromatographyto separate this protein into four toxin complexes (tc) termed, Tca,Tcb, Tcc, and Tcd encoded by the tc loci (Bowen et al., 1998).Waterfield et al. (2001) analyzed recombinant expression of the tc genesin E. coli to understand oral toxicity of Tc proteins. They found thatwithout tccC-like homologs, they could not recover oral toxicity in E.coli. These authors concluded that TccC is involved in activation oftoxin secretion. Furthermore, an amino acid sequence analysis revealedTccC and TccC-like proteins have a highly conserved core and highlyvariable extension. This structure bears resemblance to rhs-likeelements (Waterfield N R, Bowen D J, Fetherston J D, Perry R D, andffrench-Constant, R H, 2001). This similarity suggests that TccC-likeand Rhs proteins share an ancient role in toxin mobility and activationfor the Enterobacteriaceae family (ffrench-Constant, R et al, 2003).

Another microbe, Serratia entomophila, has insecticidal activity thattargets New Zealand grass grub, Costelytra zealandica, and causes amberdisease (Grimont et al., 1988). The virulence of S. entomophila islinked to a large plasmid called amber disease-associated plasmid(pADAP) (Glare et al., 1993). Hurst et al. analyzed the mutagenesis andthe nucleotide sequence of pADAP to understand how it conferspathogenicity to grass grub. They found that pADAP encodes three genesresponsible for the symptoms of amber disease, sepA, sepB, and sepC. Allthree genes are required for pathogenicity because a mutation in thesegenes abolishes amber disease. They illustrated that proteins encoded bythe sep genes are similar to the proteins encoded by the insecticidaltoxin complexes of P. luminescens. For example, the first 680 aminoacids of SepC and TccC show a strong similarity. Furthermore, thisregion resembles the rhs elements of E. coli. The sepC gene is smallerthan Rhs elements, but it encodes a hydrophilic protein core with nineRhs peptide variants. Based on the similarity between the sep and tcgenes, Hurst et al. concludes that these products are part of a newgroup of insecticidal toxins (Hurst et al., 2000).

Harada et al. discovered that, Pantoea stewartii ssp. DC283 is anaggressive pathogen that infects aphids (Harada et al., 1996). The aphidingests the bacterium and DC283 is able to aggregate in the gut andcause death of the aphid. Stavrinides et al. performed a mutagenesisscreen and discovered that the ucp1 (you cannot pass) locus isresponsible for the virulence of DC283. Analysis of the ucp1 genesequence revealed similarities to the Rhs protein family. ucp1 gene issmaller than the genes encoding RHS/YD proteins and does not have aligand binding YD repeat, but it has conserved 5′-cores, non-homologous3′ ends, and it is a membrane bound protein. These structuralsimilarities suggest enteric plant colonizers have the genetic abilityto colonize insect hosts. Furthermore, the similarities between the ucp1and rhs genes suggest that rhs-like genes have potential insecticidalactivity (Stavrinides et al., 2010).

Despite these known protein, it is possible that insects may evolveresistance to plants expressing these known genes. Accordingly, there isa need to find novel proteins that have insecticidal activities.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides the nucleotide sequenceof insecticidal proteins from bacterium Chromobacterium subtsugae.Isolation and partial characterization of this bacterium is described,for example, in U.S. Pat. No. 7,244,607. Additionally provided are aminoacid sequences of polypeptides encoded by the Chromobacterium subtsugaeinsecidal proteins.

In another aspect, the present disclosure provides isolated nucleicacids (e.g., DNA, RNA, nucleic acid analogues) comprising C. subtsugaeinsecidal proteins sequences, gene sequences, fragments thereof, and ormutant variants. Also provided are nucleic acid vectors (e.g., plasmidvectors, viral vectors), including expression vectors, comprisingnucleic acids having C. subtsugae gene sequences, and/or fragmentsthereof. Exemplary bacterial vectors include, but are not limited to,Agrobacterium tumefaciens, Rhizobium sp. NGR234, Sinorhizobium meliloti,and Mesorhizobium loti.

Exemplary viral vectors include, but are not limited to, cauliflowermosaic virus (CaMV), pea early browning virus (PEBV), bean pod mottlevirus (BPMV), cucumber mosaic virus (CMV), apple latent spherical virus(ALSV), tobacco mosaic virus (TMV), potato virus X, brome mosaic virus(BMV) and barley stripe mosaic virus (BSMV).

Cells transfected with the foregoing nucleic acids or vectors are alsoprovided. Such cells can be plant cells, insect cells, mammalian cells,bacterial cells, or fungal cells (e.g., yeast). Plants comprising cells(plant or otherwise) that have been transfected with the foregoingnucleic acids or vectors, seeds from said plants, and the progeny ofsaid plants are also provided. Transfected bacterial cells can includeAgrobacteria (e.g., Agrobacterium tumefaciens), Rhizobium, Sinorhizobiummeliloti, and Mesorhizobium loti. Insect vectors (e.g., Homalodiscavitripennis, the glassy-winged sharpshooter) comprising nucleic acidvectors which themselves comprise C. subtsugae sequences, are alsoprovided.

In additional embodiments, polypeptides encoded by the C. subtsugaegenes are provided. Functional fragments of C. subtsugae polypeptides,and conservatively substituted variants of C. subtsugae polypeptides,are also provided.

In further embodiments, plants comprising one or more isolated nucleicacids comprising C. subtsugae gene sequences and/or fragments thereofare provided. These isolated nucleic acids can be present on theexterior of the plant or internally within or between cells.

In additional embodiments, plants comprising one or more nucleic acidvectors, wherein said vector or vectors comprise C. subtsugae genesequences and/or fragments thereof, are provided. Said vectors can bepresent on the exterior of the plant or internally.

In yet additional embodiments, plants comprising one or more C.subtsugae polypeptides are provided. Said C. subtsugae polypeptides canbe present on the exterior of the plant or internally.

Also provided are plants comprising one or more functional fragmentsand/or one or more conservatively substituted variants of a C. subtsugaepolypeptide or polypeptides. Said fragments and/or conservativelysubstituted variants can be present on the exterior of the plant orinternally.

Progeny of the aforementioned plants are also provided. In addition,seeds from the aforementioned plants, and from their progeny, areprovided.

Also disclosed herein are methods for controlling pests; e.g., methodsfor modulating pest infestation in a plant. Such pests can be, forexample, insects, fungi, nematodes, mites, moths or aphids. The methodsinclude application of a nucleic acid comprising a C. subtsugae genesequence or fragment thereof to a plant, either internally orexternally. Additional methods include application of a C. subtsugaepolypeptide, or fragment thereof, or conservatively substituted variantthereof, to a plant, either internally or externally.

Also provided are pesticidal (e.g., insecticidal) compositionscomprising nucleic acids and/or polypeptides encoded by the C. subtsugaegenes. Such compositions can optionally include other insecticides orpesticides, either naturally-occurring or man-made.

Disclosed herein, inter alia, are the following embodiments:

1. A cell comprising a recombinant vector having a heterologous promoteroperately linked to a nucleotide encoding a polypeptide with 95%identity to SEQ ID NOs: 1-3.

2. A plant or plant parts comprising one or more cells according toembodiment 1 or progeny or seed thereof.

3. The plant or plant parts of embodiment 2, wherein said plant part isselected from the group consisting of pollen, ovule, flower, shoot,root, stalk, silk, tassel, ear, and leaf tissue.

5. An antibody that binds to the polypeptide of embodiment 1.

6. The cell of embodiment 1, wherein said cell is a bacterial,mammalian, or fungal cell.

7. A method of producing an insect resistant plant comprising the stepof transforming the recombinant vector of embodiment 1 into a plantcell.

8. Anti-counterfeit milled seed having, as an indication of origin, aplant cell of embodiment 1.

9. A pesticidal composition comprising (a) one or more nucleic acidsand/or polypeptides having a sequence as set forth in SEQ ID NOS:1-6 and(b) a carrier.

10. The pesticidal composition of embodiment 9, wherein the compositionis an insecticide.

11. The pesticidal composition of embodiment 10, further comprising asecond pesticide.

12. The pesticidal composition of embodiment 11, wherein the secondpesticide is an insecticide.

13. A method for modulating pest infestation in a plant, the methodcomprising contacting a plant or a plant part with an amount of thepesticidal composition according to embodiment 9 effective to modulatesaid pest infestation.

14. The method of embodiment 13, wherein the pest is selected from thegroup consisting of insects, fungi, nematodes, bacteria and mites.

15. The method of embodiment 14, wherein the insects comprise cabbageloopers, Beet armyworms, lygus, or Diamondback Moth.

16. A seed coating material comprising the polypeptide of embodiment 1,and one or more of a carrier, diluent, or adjuvant.

DESCRIPTION OF THE INVENTION

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields ofagriculture, plant molecular biology, entomology, cell biology,molecular biology, biochemistry, recombinant DNA and related fields asare within the skill of the art. Such techniques are described in theliterature and thereby available to those of skill in the art. See, forexample, Alberts, B. et al., “Molecular Biology of the Cell,” 5^(th)edition, Garland Science, New York, N.Y., 2008; Voet, D. et al.“Fundamentals of Biochemistry: Life at the Molecular Level,” 3^(rd)edition, John Wiley & Sons, Hoboken, N.J., 2008; Sambrook, J. et al.,“Molecular Cloning: A Laboratory Manual,” 3^(rd) edition, Cold SpringHarbor Laboratory Press, 2001; Ausubel, F. et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, New York, 1987 and periodicupdates; Glover, DNA Cloning: A Practical Approach, volumes I and II,IRL Press (1985), volume III, IRL Press (1987); Perbal, A PracticalGuide to Molecular Cloning, John Wiley & Sons (1984); Rigby (ed.), Theseries “Genetic Engineering” (Academic Press); Setlow & Hollaender(eds.), The series “Genetic Engineering: Principles and Methods,” PlenumPress; Gait (ed.), Oligonucleotide Synthesis: A Practical Approach, IRLPress (1984, 1985); Eckstein (ed.) Oligonucleotides and Analogues: APractical Approach, IRL Press (1991); Hames & Higgins, Nucleic AcidHybridization: A Practical Approach, IRL Press (1985); Hames & Higgins,Transcription and Translation: A Practical Approach, IRL Press (1984);B. Buchanan, W. Gruissem & R. Jones (eds.) “Biochemistry and MolecularBiology of Plants,” Wiley (2002) and the series “Methods in Enzymology,”Academic Press, San Diego, Calif. The disclosures of all of theforegoing references are incorporated by reference in their entiretiesfor the purpose of describing methods and compositions in the relevantarts.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is included therein. Smaller ranges are also included. Theupper and lower limits of these smaller ranges are also includedtherein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” and “the” include plural references unless thecontext clearly dictates otherwise.

As used herein, the term “construct” means any recombinantpolynucleotide molecule such as a plasmid, cosmid, virus, autonomouslyreplicating polynucleotide molecule, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA polynucleotide molecule,derived from any source, capable of genomic integration or autonomousreplication, comprising a polynucleotide molecule where one or morepolynucleotide molecule has been linked in a functionally operativemanner, i.e., operably linked.

As used herein, the term “operably linked” refers to a first moleculejoined to a second molecule, wherein the molecules are so arranged thatthe first molecule affects the function of the second molecule. The twomolecules may or may not be part of a single contiguous molecule and mayor may not be adjacent. For example, a promoter is operably linked to atranscribable polynucleotide molecule if the promoter modulatestranscription of the transcribable polynucleotide molecule of interestin a cell.

Constructs may include any promoter or leader known in the art. Forexample, a promoter may be operably linked to a heterologousnon-translated 5′ leader such as one derived from a heat shock proteingene (see, for example, U.S. Pat. Nos. 5,659,122 and 5,362,865).Alternatively, a leader may be operably linked to a heterologouspromoter such as the Cauliflower Mosaic Virus 35S transcript promoter(see, U.S. Pat. No. 5,352,605).

As used herein, the term “transcribable polynucleotide molecule” refersto any DNA molecule capable of being transcribed into a RNA molecule,including, but not limited to, those having protein coding sequences(SEQ ID NOs:4-6) and those having sequences useful for gene suppression.A “transgene” refers to a transcribable polynucleotide moleculeheterologous to a host cell and/or a transcribable polynucleotidemolecule artificially incorporated into a host cell's genome.

A promoter may be operably linked to a transcribable polynucleotidemolecule that is heterologous with respect to the promoter molecule. Asused herein, the term “heterologous” refers to the combination of two ormore polynucleotide molecules when such a combination would not normallybe found in nature. For example, the two molecules may be derived fromdifferent species and/or the two molecules may be derived from differentgenes, e.g. different genes from the same species or the same genes fromdifferent species. A promoter is thus heterologous with respect to anoperably linked transcribable polynucleotide molecule if such acombination is not normally found in nature, i.e. that transcribablepolynucleotide molecule is not naturally occurring operably linked incombination with that promoter molecule.

The transcribable polynucleotide molecule may generally be any DNAmolecule for which expression of an RNA transcript is desired. Suchexpression of an RNA transcript may result in translation of theresulting mRNA molecule and thus protein expression. Alternatively, atranscribable polynucleotide molecule may be designed to ultimatelycause decreased expression of a specific gene or protein that canenhance protein expression of SEQ ID NOs: 1-3. This may be accomplishedby using a transcribable polynucleotide molecule that is oriented in theantisense direction. One of ordinary skill in the art is familiar withusing such antisense technology. Briefly, as the antisense transcribablepolynucleotide molecule is transcribed, the RNA product hybridizes toand sequesters a complementary RNA molecule inside the cell. This duplexRNA molecule cannot be translated into a protein by the cell'stranslational machinery and is degraded in the cell. Any gene may benegatively regulated in this manner.

Polynucleotides and Oligonucleotides

A polynucleotide is a polymer of nucleotides, and the term is meant toembrace smaller polynucleotides (fragments) generated by fragmentationof larger polynucleotides. The terms polynucleotide and nucleic acidencompass both RNA and DNA, as well as single-stranded anddouble-stranded polynucleotides and nucleic acids. Polynucleotides alsoinclude modified polynucleotides and nucleic acids, containing suchmodifications of the base, sugar or phosphate groups as are known in theart.

An oligonucleotide is a short nucleic acid, generally DNA and generallysingle-stranded. Generally, an oligonucleotide will be shorter than 200nucleotides, more particularly, shorter than 100 nucleotides, mostparticularly, 50 nucleotides or shorter.

Modified bases and base analogues, e.g., those able to form Hoogsteenand reverse Hoogsteen base pairs with the naturally-occurring bases, areknown in the art. Examples include, but are not limited to,8-oxo-adenosine, pseudoisocytidine, 5-methyl cytidine, inosine,2-aminopurine and various pyrrolo- and pyrazolopyrimidine derivatives.Similarly, modified sugar residues or analogues, for example2′-O-methylribose or peptide nucleic acid backbones, can also form acomponent of a modified base or base analogue. See, for example, Sun andHelene (1993) Curr. Opin. Struct. Biol. 3:345-356. Non-nucleotidemacromolecules capable of any type of sequence-specific interaction witha polynucleotide are useful in the methods and compositions disclosedherein. Examples include, but are not limited to, peptide nucleic acids,minor groove-binding agents and antibiotics. New modified bases, baseanalogues, modified sugars, sugar analogues, modified phosphates andphosphate analogues capable of participating in duplex or triplexformation are available in the art, and are useful in the methods andcompositions disclosed herein.

Homology and Identity of Nucleic Acids and Polypeptides

“Homology” or “identity” or “similarity” as used herein in the contextof nucleic acids and polypeptides refers to the relationship between twopolypeptides or two nucleic acid molecules based on an alignment of theamino acid sequences or nucleic acid sequences, respectively. Homologyand identity can each be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. For example, a“reference sequence” can be compared with a “test sequence.” When aposition in the reference sequence is occupied by the same base or aminoacid at an equivalent position in the test sequence, then the moleculesare identical at that position; when the equivalent position is occupiedby a similar amino acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. The relatedness of two sequences, whenexpressed as a percentage of homology/similarity or identity, is afunction of the number of identical or similar amino acids at positionsshared by the sequences being compared. In comparing two sequences, theabsence of residues (amino acids or nucleic acids) or presence of extraresidues, in one sequence as compared to the other, also decreases theidentity and homology/similarity.

As used herein, the term “identity” refers to the percentage ofidentical nucleotide or amino acid residues at corresponding positionsin two or more sequences when the sequences are aligned to maximizesequence matching, i.e., taking into account gaps and insertions.Identity can be readily calculated by known methods, including but notlimited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988). Methods to determine identity aredesigned to give the highest degree of match between the sequencestested. Moreover, methods to determine identity are codified in publiclyavailable computer programs. Computer program methods to determineidentity between two sequences include, but are not limited to, the GCGprogram package (Devereux et al. (1984) Nucleic Acids Research 12:387),BLASTP, BLASTN, and FASTA (Altschul et al. (1990) J. Molec. Biol.215:403-410; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).The BLAST X program is publicly available from NCBI and other sources.See, e.g., BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda,Md. 20894; Altschul et al. (1990) J. Mol. Biol. 215:403-410. The wellknown Smith-Waterman algorithm can also be used to determine identity.

For sequence comparison, typically one sequence acts as a referencesequence, to which one or more test sequences are compared. Sequencesare generally aligned for maximum correspondence over a designatedregion, e.g., a region at least about 20, 25, 30, 35, 40, 45, 50, 55,60, 65 or more amino acids or nucleotides in length, and the region canbe as long as the full-length of the reference amino acid sequence orreference nucleotide sequence. When using a sequence comparisonalgorithm, test and reference sequences are input into a computerprogram, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Examples of algorithms that are suitable for determining percentsequence identity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (1990) J. Mol. Biol. 215:403-410 andAltschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information at www.ncbi.nlm.nih.gov.Further exemplary algorithms include ClustalW (Higgins et al. (1994)Nucleic Acids Res. 22:4673-4680), available atwww.ebi.ac.uk/Tools/clustalw/index.html.

In another embodiment, sequence identity between two nucleic acids canalso be described in terms of annealing, reassociation, or hybridizationof two polynucleotides to each other, mediated by base-pairing.Hybridization between polynucleotides proceeds according to known andart-recognized base-pairing properties, such that adenine base-pairswith thymine or uracil, and guanine base-pairs with cytosine. Theproperty of a nucleotide that allows it to base-pair with a secondnucleotide is called complementarity. Thus, adenine is complementary toboth thymine and uracil, and vice versa; similarly, guanine iscomplementary to cytosine and vice versa. An oligonucleotide orpolynucleotide which is complementary along its entire length with atarget sequence is said to be perfectly complementary, perfectlymatched, or fully complementary to the target sequence, and vice versa.Two polynucleotides can have related sequences, wherein the majority ofbases in the two sequences are complementary, but one or more bases arenoncomplementary, or mismatched. In such a case, the sequences can besaid to be substantially complementary to one another. If twopolynucleotide sequences are such that they are complementary at allnucleotide positions except one, the sequences have a single nucleotidemismatch with respect to each other.

The term “substantially identical” refers to identity between a firstamino acid sequence that contains a sufficient or minimum number ofamino acid residues that are i) identical to, or ii) conservativesubstitutions of, aligned amino acid residues in a second amino acidsequence such that the first and second amino acid sequences share acommon structural domain and/or common functional activity. For example,amino acid sequences that contain a common structural domain having atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%identity to an amino acid sequence as disclosed herein (i.e., SEQ IDNOs:1-3) are termed substantially identical. In the context ofnucleotide sequence, the term “substantially identical” is used hereinto refer to a first nucleic acid sequence that contains a sufficient orminimum number of nucleotides that are identical to aligned nucleotidesin a second nucleic acid sequence such that the first and secondnucleotide sequences encode a polypeptide having common functional orstructural activity, or encode a common structural polypeptide domain ora common functional polypeptide activity.

In another embodiment, the term “homology” describes a mathematicallybased comparison of sequence similarities which is used to identifygenes or proteins with similar functions or motifs. A referencenucleotide or amino acid sequence (e.g., a sequence as disclosed herein)is used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members, relatedsequences or homologues. Such searches can be performed using the NBLASTand XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol.Biol. 215:403-410. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to a reference nucleotide sequence. BLAST amino acid searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to a reference amino acidsequence. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402. When utilizing the BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and BLAST) can be used (see the world wide web at:ncbi.nlm.nih.gov).

Nucleic acids and polynucleotides of the present disclosure encompassthose having an nucleotide sequence that is at least 75%, at least 80%,at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%or 100% identical to any of SEQ ID NOs:4-6.

Nucleotide analogues and amino acid analogues are known in the art.Accordingly, nucleic acids (i.e., SEQ ID NOs:4-6) comprising nucleotideanalogues and polypeptides (i.e., SEQ ID NOs:1-3) comprising amino acidanalogues are also encompassed by the present disclosure.

Transcribable polynucleotide molecules can be genes of agronomicinterest. As used herein, the term “gene of agronomic interest” refersto a transcribable polynucleotide molecule that when expressed in aparticular plant tissue, cell, or cell type provides a desirablecharacteristic associated with plant morphology, physiology, growth,development, yield, product, nutritional profile, disease or insect/pestresistance, and/or environmental or chemical tolerance. Genes ofagronomic interest include, but are not limited to, insect control genesencoded by SEQ ID NOs: 4-6 or its associated protein SEQ ID NOs:1-3,those encoding a yield protein, a stress resistance protein, adevelopmental control protein, a tissue differentiation protein, ameristem protein, an environmentally responsive protein, a senescenceprotein, a hormone responsive protein, an abscission protein, a sourceprotein, a sink protein, a flower control protein, a seed protein, anherbicide resistance protein, a disease resistance protein, a fatty acidbiosynthetic enzyme, a tocopherol biosynthetic enzyme, an amino acidbiosynthetic enzyme, a pesticidal protein, or any other agent such as anantisense or RNAi molecule targeting a particular gene for suppressionto enhance protein expressions of SEQ ID NO: 1-3. The product of a geneof agronomic interest can act within the plant in order to cause aneffect upon the plant physiology or metabolism or can be act as apesticidal agent in the diet of a pest that feeds on the plant.

As used herein, “control plant” means a plant that does not contain therecombinant DNA that expressed a protein which imparts an enhancedtrait. A control plant is to identify and select a plant that has anenhance trait. A suitable control plant can be a non-transgenic plant ofthe parental line used to generate a transgenic plant, e.g., devoid ofrecombinant DNA. A suitable control plant can in some cases be a progenyof a hemizygous transgenic plant line that is does not contain therecombinant DNA, known as a negative segregant.

As used herein, an “enhanced trait” means a characteristic of atransgenic plant that includes, but is not limited to, an enhanceagronomic trait characterized by enhanced insect resistance, enhancedplant morphology, physiology, growth and development, yield, nutritionalenhancement, disease or pest resistance, or environmental or chemicaltolerance. In more specific aspects of this invention enhanced trait isselected from group of enhanced traits consisting of enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil. In anaspect of the invention the enhanced trait is enhanced yield includingincreased yield under non-stress conditions and increased yield underenvironmental stress conditions. Stress conditions can include, forexample, drought, shade, fungal disease, viral disease, bacterialdisease, insect infestation, nematode infestation, cold temperatureexposure, heat exposure, osmotic stress, reduced nitrogen nutrientavailability, reduced phosphorus nutrient availability and high plantdensity. “Yield” can be affected by many properties including withoutlimitation, plant height, pod number, pod position on the plant, numberof internodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Yield can also be affected by efficiency ofgermination (including germination in stressed conditions), growth rate(including growth rate in stressed conditions), ear number, seed numberper ear, seed size, composition of seed (starch, oil, protein) andcharacteristics of seed fill.

Increased yield of a plant can be measured in a number of ways,including test weight, seed number per plant, seed weight, seed numberper unit area (e.g., seeds, or weight of seeds, per acre), bushels peracre, tonnes per acre, tons per acre, kilo per hectare. For example,maize yield can be measured as production of shelled corn kernels perunit of production area in bushels per acre or metric tons per hectare,often reported on a moisture adjusted basis at about 15.5 percentmoisture. Increased yield can result from improved utilization of keybiochemical compounds such as nitrogen, phosphorous and carbohydrate, orfrom improved responses to environmental stresses, such as cold, heat,drought, salt, and attack by pests or pathogens.

Conservative Substitutions and Functional Fragments

In comparing amino acid sequences, residue positions which are notidentical can differ by conservative amino acid substitutions.Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. With respect to a referencepolypeptide sequence, a test polypeptide sequence that differs only byconservative substitutions is denoted a “conservatively substitutedvariant” of the reference sequence.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are known in the art. Similarly,methods for determining protein function are known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. See Ausubel et al., supra. The ability of a protein to interactwith another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, eithergenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245 246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

Typically, a functional fragment retains at least 50% of the activity orfunction of the polypeptide. In some embodiments, a functional fragmentretains at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99% or 100% of the activity or function of the polypeptide.

A functional fragment of a polypeptide can include conservative aminoacid substitutions (with respect to the native polypeptide sequence)that do not substantially alter the activity or function of thepolypeptide. The term “conservative amino acid substitution” refers togrouping of amino acids on the basis of certain common structures and/orproperties. With respect to common structures, amino acids can begrouped into those with non-polar side chains (glycine, alanine, valine,leucine, isoleucine, methionine, proline, phenylalanine and tryptophan),those with uncharged polar side chains (serine, threonine, asparagine,glutamine, tyrosine and cysteine) and those with charged polar sidechains (lysine, arginine, aspartic acid, glutamic acid and histidine). Agroup of amino acids containing aromatic side chains includesphenylalanine, tryptophan and tyrosine. Heterocyclic side chains arepresent in proline, tryptophan and histidine. Within the group of aminoacids containing non-polar side chains, those with short hydrocarbonside chains (glycine, alanine, valine. leucine, isoleucine) can bedistinguished from those with longer, non-hydrocarbon side chains(methionine, proline, phenylalanine, tryptophan). Within the group ofamino acids with charged polar side chains, the acidic amino acids(aspartic acid, glutamic acid) can be distinguished from those withbasic side chains (lysine, arginine and histidine).

A functional method for defining common properties of individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz, G. E.and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag,1979). According to such analyses, groups of amino acids can be definedin which amino acids within a group are preferentially substituted forone another in homologous proteins, and therefore have similar impact onoverall protein structure (Schulz, G. E. and R. H. Schirmer, supra).According to this type of analysis, conservative amino acidsubstitution” refers to a substitution of one amino acid residue foranother sharing chemical and physical properties of the amino acid sidechain (e.g., charge, size, hydrophobicity/hydrophilicity). Following areexamples of amino acid residues sharing certain chemical and/or physicalproperties:

(i) amino acids containing a charged group, consisting of Glu, Asp, Lys,Arg and His,

(ii) amino acids containing a positively-charged group, consisting ofLys, Arg and His,

(iii) amino acids containing a negatively-charged group, consisting ofGlu and Asp,

(iv) amino acids containing an aromatic group, consisting of Phe, Tyrand Trp,

(v) amino acids containing a nitrogen ring group, consisting of His andTrp,

(vi) amino acids containing a large aliphatic non-polar group,consisting of Val, Leu and Ile,

(vii) amino acids containing a slightly-polar group, consisting of Metand Cys,

(viii) amino acids containing a small-residue group, consisting of Ser,Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro,

(ix) amino acids containing an aliphatic group consisting of Val, Leu,Ile, Met and Cys, and

(x) amino acids containing a hydroxyl group consisting of Ser and Thr.

Certain “conservative substitutions” may include substitution within thefollowing groups of amino acid residues: gly, ala; val, ile, leu; asp,glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

Thus, as exemplified above, conservative substitutions of amino acidsare known to those of skill in this art and can be made generallywithout altering the biological activity or function of the resultingmolecule. Those of skill in this art also recognize that, in general,single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity. See, e.g.,Watson, et al., “Molecular Biology of the Gene,” 4th Edition, 1987, TheBenjamin/Cummings Pub. Co., Menlo Park, Calif., p. 224.

Polypeptides of the present disclosure encompass those having 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 or more amino acid substitutions compared to anamino acid sequence as set forth in SEQ ID NOs:1-3, e.g., conservativeamino acid substitutions. Amino acid residues that can be substitutedcan be located at residue positions that are not highly conserved. Theordinarily skilled artisan will appreciate that, based on location ofthe active sites and/or on homology to related proteins, a protein willtolerate substitutions, deletions, and/or insertions at certain of itsamino acid residues, without significant change in its overall physicaland chemical properties.

Polypeptides of the present disclosure encompass those having an aminoacid sequence that is at least 75%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% identical to any of the polypeptides shown in SEQ IDNOs:1-3.

RNA Suppression

Small RNAs that regulate protein expression include miRNAs andta-siRNAs. A miRNA is a small (typically about 21 nucleotide) RNA thathas the ability to modulate the expression of a target gene by bindingto messenger RNA for the target protein leading to destabilization ofthe target protein messenger RNA or translational inhibition of thetarget protein messenger RNA, ultimately resulting in reduction of thetarget protein. The design and construction of ta-siRNA constructs andtheir use in the modulation of protein in transgenic plant cells isdisclosed by Allen and Carrington in US Patent Application PublicationUS 2006/0174380 A1 which is incorporated herein by reference. Theexpression or suppression of such small RNAs are aspects of theinvention that are conveniently illustrated by reference to use ofmiRNAs.

Recombinant DNA constructs can be used to modify the activity of nativemiRNAs by a variety of means. By increasing the expression of a miRNA,e.g. temporally or spatially, the modulation of expression of a nativetarget gene can be enhanced. An alternative gene suppression approachfor suppressing the expression of a target protein can include the useof a recombinant DNA construct that produces a synthetic miRNA that isdesigned to bind to a native or synthetic miRNA recognition site onmessenger RNA for the target protein.

By reducing the expression of a miRNA, the modulation of a native targetgene can be diminished resulting in enhanced expression of the targetprotein, such as SEQ ID NOs: 1-3. More specifically, the expression of atarget protein can be enhanced by suppression of the activity of themiRNA that binds to a recognition site in the messenger RNA that istranscribed from the native gene for the target protein. Several typesof recombinant DNA constructs can be designed to suppress the activityof a miRNA.

For example, a recombinant DNA construct that produces an abundance ofRNA with the miRNA recognition site can be used as a decoy for thenative miRNA allowing endogenous messenger RNA with the miRNArecognition site to be translated to the target protein withoutinterference from native miRNA. A recombinant DNA construct thatproduces RNA with a modified miRNA recognition site, e.g. withnucleotides at positions 10 and/or 11 in a 21 mer miRNA recognition sitewhich are unpaired with respect to the native miRNA, can be used tosequester natively expressed miRNA thereby reducing the cleavage thatnormally occurs when miRNA binds to a recognition site. The unpairednucleotides can be produced e.g. through additional nucleotides betweenpositions 10 and 11 or through substitutions of the nucleotides atpositions 10 and 11.

Additionally, a recombinant DNA construct can be created that producesRNA that can be processed in plants into synthetic small RNA(miRNA-like) that can bind endogenous miRNA recognition sites but isunable to induce cleavage of mRNA because the small RNA is modified, forinstance by having a modified nucleotide at positions 10 and/or 11 or adeletion that produces a bulge between positions 10 and 11 when thesmall RNA is paired with the miRNA recognition site. The resultingsynthetic small RNA, a cleavage blocker, can reduce endogenous miRNAbinding and thus block cleavage of a protected miRNA target siteenhancing the expression of a target protein.

A recombinant DNA construct designed for producing a modified messengerRNA for the protein where the native miRNA recognition site is modifiedto be resistant to the binding of cognate miRNA which regulates thenative gene can also be used to express protein from heterologousmessenger RNA that is no longer modulated by the native miRNA.

The activity of a miRNA which down-regulates an endogenous protein isenhanced by enhancing the expression of the miRNA or by enhancing theability of the miRNA to bind an RNA encoding the target protein. Arecombinant DNA encoding an RNA encoding the miRNA or a miRNA-sensitivemessenger RNA encoding the protein in which a miRNA binding site isadded are designed to enhance miRNA activity resulting in enhancedsuppression of the target mRNA and cognate protein. Recombinant DNAencoding an RNA encoding a miRNA, or a miRNA-sensitive RNA are designedusing methods disclosed in US Patent Application Publication US2009/0070898 A1.

Some, if not many, miRNAs modulate the expression of multiple proteinsor biochemical pathways. Plants can be provided with enhanced traits notso much from the suppression or enhancement of the expression of aparticular protein, as from change of enzyme activity in a pathway bymodulating the level of a miRNA. Thus, aspects of this invention areachieved by enhanced miRNA activity resulting from use in plant cells ofrecombinant DNA constructs that produce an enhanced level of a miRNA.Other aspects of this invention are achieved by reduced miRNA activityresulting from use in transgenic plant cells of recombinant DNAconstructs that produce a reduced level or activity of a miRNA.

C. subtsugae Nucleic Acids

Also provided are nucleotide sequences encoding C. subtsugae genes andnucleotide sequences of functional RNA molecules (e.g., rRNAs, tRNAs)(SEQ ID NOs:4-6). Nucleic acids comprising these sequences are alsoprovided. Fragments of C. subtsugae gene sequences are also provided.Such fragments are 10 or more, 25 or more, 50 or more, 75 or more, 100or more 200 or more, 500 or more, or 1,000 or more nucleotides inlength. Nucleic acids having a sequence that is 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to theaforementioned sequences are also provided. The nucleic acids disclosedherein can be either DNA or RNA, and can be either single-stranded ordouble-stranded. Nucleic acids comprising nucleotide sequences that arecomplementary to the aforementioned sequences are also provided, as arenucleic acids that hybridize to the aforementioned nucleic acids understringent conditions.

The present disclosure also provides polynucleotides comprising anucleotide sequence encoding any of the polypeptide sequences disclosedherein. Such a polynucleotide has a nucleotide sequence that is at least70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, at least 99%, at least 99.5% or 100%) identicalto a contiguous sequence of a nucleic acid that encodes any of thepolypeptides disclosed herein. The percentage identity is based on theshorter of the sequences compared. Known programs such as BLASTN (2.0.8)(Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402) using defaultparameters and no filter can be employed to make a sequence comparison.Nucleic acid sequence identity (e.g. between two differentpolynucleotides encoding identical amino acid sequences) can be lowerthan the percent of amino acid sequence identity due to degeneracy ofthe genetic code.

Examples of nucleic acid sequences in a polynucleotide encoding apolypeptide of the present disclosure can be found among SEQ ID NOs:4-6.These nucleic acid sequences can also be provided in an expressionvector (see below).

C. subtsugae Polypeptides and Proteins

The present disclosure provides the amino acid sequences of proteinsencoded by the C. subtsugae genome, as well as polypeptides comprisingsaid amino acid sequences (i.e., SEQ ID NOs:1-3). Functional fragmentsand conservatively-substituted variants of said polypeptides are alsoprovided. In addition, fragments of the polypeptides disclosed hereinthat do not retain function are also provided and are useful, e.g., asepitopes for production of antibodies. Such fragments are 4 or more, 10or more, 25 or more, 50 or more, 75 or more, 100 or more 200 or more,500 or more, or 1,000 or more amino acids in length.

The present disclosure also provides a polypeptide comprising an aminoacid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or 99.9% identical to acontiguous sequence of a polypeptide as disclosed herein. The percentageidentity is based on the shorter of the sequences compared. Methods fordetermining degree of polypeptide sequence identity are known in theart.

The subject polypeptides can include amino acid sequences derived fromany of SEQ ID NOs:1-3 further comprising heterologous amino acidsequences. Such polypeptides can be fusion proteins, such as a fusionprotein containing epitope tags, purification tags, and/or detectablelabels. A fusion protein can optionally include a linker sequencebetween the heterologous sequences and the C. subtsugae amino acidsequence. Methods for producing fusion proteins are known in the art.Other heterologous elements and exemplary fusion proteins are describedin more detail below.

Exemplary polypeptides containing heterologous elements may include mycand/or His₆ tags and may optionally include flanking linker sequences.

Polypeptides of the present disclosure further encompass those that arejoined to a reporter polypeptide, e.g., a fluorescent protein, and/orconjugated to a molecule. The molecule conjugated to the polypeptide canbe a carrier molecule or a moiety that facilitates delivery and/orincreases the half-life of the subject polypeptide.

Polypeptides of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis). The subject polypeptide can be prepared bysolid-phase synthesis methods known in the art, (e.g., Fmoc- or t-Bocchemistry), such as those described by Merrifield (1963) J. Am. Chem.Soc. 85:2149 and Methods in Molecular Biology, Vol 35: Peptide SynthesisProtocols.

It should be noted that the polypeptides of the present disclosure canalso contain additional elements, such as a detectable label, e.g., aradioactive label, a fluorescent label, a biotin label, animmunologically detectable label (e.g., a hemagglutinin (HA) tag, apoly-Histidine tag) and the like. Additional elements can be provided(e.g., in the form of fusion polypeptides) to facilitate expression(e.g. N-terminal methionine and/or a heterologous signal sequence tofacilitate expression in host cells), and/or isolation (e.g., biotintag, immunologically detectable tag) of the polypeptides of thedisclosure through various methods. The polypeptides can also optionallybe immobilized on a support through covalent or non-covalent attachment.

Isolation and purification of the subject polypeptides can beaccomplished according to methods known in the art. The term “isolated”is intended to mean that a compound (e.g. polypeptide or polynucleotide)is separated from all or some of the components that accompany it innature. “Isolated” also refers to the state of a compound separated fromall or some of the components that accompany it during manufacture(e.g., chemical synthesis, recombinant expression, culture medium, andthe like).

For example, a polypeptide according to the present disclosure can beisolated from a lysate of cells that have been genetically modified toexpress the subject polypeptide, from a cell culture medium, or from asynthetic reaction mixture. Isolation can additionally be achieved byimmunoaffinity purification, which generally involves contacting asample with an antibody (optionally immobilized) that specifically bindsto an epitope of the polypeptide, washing to remove non-specificallybound material, and eluting specifically bound polypeptide. Isolatedpolypeptide can be further purified by dialysis and other methodsnormally employed in protein purification, e.g. metal chelatechromatography, ion-exchange, and size exclusion.

Homologues

In yet another embodiment, the present disclosure provides methods ofobtaining homologues of the fragments of the C. subtsugae genesdisclosed herein, and homologues of the proteins encoded by the ORFsdisclosed herein. Specifically, by using the nucleotide and amino acidsequences disclosed herein as a probe or as primers, and techniques suchas PCR cloning and colony/plaque hybridization, one skilled in the artcan obtain said homologues. Such homologues can be obtained from anyorganism; e.g., other species of Chromobacterium or other bacteria.

In another embodiment, homologs can be identified by comparison of aminoacid sequence, e.g. manually or by use of a computer-based tool usingknown homology-based search algorithms such as those commonly known andreferred to as BLAST, FASTA, and Smith-Waterman. A local sequencealignment program, e.g. BLAST, can be used to search a database ofsequences to find similar sequences, and the summary Expectation value(E-value) used to measure the sequence base similarity. Because aprotein hit with the best E-value for a particular organism may notnecessarily be an ortholog, e.g., have the same function, or be the onlyortholog, a reciprocal query is used to filter hit sequences withsignificant E-values for ortholog identification. The reciprocal queryentails search of the significant hits against a database of amino acidsequences from the base organism that are similar to the sequence of thequery protein. A hit can be identified as an ortholog, when thereciprocal query's best hit is the query protein itself or a proteinencoded by a duplicated gene after speciation. A further aspect of thehomologs encoded by DNA useful in the transgenic plants of the inventionare those proteins that differ from a disclosed protein as the result ofdeletion or insertion of one or more amino acids in a native sequence.

Antibodies, Detection Methods, Kits

Also provided are antibodies which selectively bind a protein orpolypeptide fragment encoded by the C. subtsugae genes such as SEQ IDNOs:1-3. Such antibodies, in addition, can comprise a detectable labeland/or be attached to a solid support. Such antibodies include bothmonoclonal and polyclonal antibodies. Also provided are hybridomas whichproduce the above-described monoclonal antibodies.

In additional embodiments, the present disclosure provides methods ofidentifying test samples derived from cells that express one or more ofthe ORFs disclosed herein, or homologues thereof. Such methods compriseincubating a test sample with one or more of the antibodies of thepresent disclosure, or one or more fragments of the C. subtsugae genes,under conditions which allow a skilled artisan to determine if thesample contains the ORF (or portion thereof) or product producedtherefrom.

In additional embodiments, kits are provided which contain the necessaryreagents to carry out the above-described assays. Specifically, providedherein is a compartmentalized kit designed to receive, in closeconfinement, one or more containers which comprises: (a) a firstcontainer comprising one of the antibodies, or one of the C. subtsugaegene fragments of the present disclosure; and (b) one or more othercontainers comprising one or more of the following: wash reagents,reagents capable of detecting presence of bound antibodies or reagentscapable of detecting presence of hybridized nucleic acids.

Using the isolated proteins disclosed herein, the present disclosurefurther provides methods of obtaining and identifying agents capable ofbinding to a protein encoded by a C. subtsugae ORF. Specifically, suchagents include antibodies (described above), peptides, carbohydrates,pharmaceutical agents and the like. Such methods comprise the steps of:(a) contacting an agent with an isolated protein encoded by one of theORFs disclosed herein; and (b) determining whether the agent binds tosaid protein. Methods for detecting protein-protein binding are known inthe art and include, for example, filter-binding, immunoprecipitation,two-hybrid assays, gel retardation and reporter subunit complementation.See, for example, U.S. Pat. Nos. 5,503,977 and 5,585,245; Fields et al.(1989) Nature 340:245-247; Bai et al. (1996) Meth. Enzymol. 273:331-347and Luo et al. (1997) BioTechniques 22:350-352.

Vectors

For embodiments in which a polypeptide is produced using recombinanttechniques, the methods can involve any suitable construct and anysuitable host cell, which can be a prokaryotic or eukaryotic cell (e.g.a bacterial host cell, a yeast host cell, a plant host cell, an insecthost cell, or a cultured mammalian host cell). Methods for introducinggenetic material into host cells are known in the art and include, forexample, biolistics, transformation, electroporation, lipofection,conjugation, calcium phosphate co-precipitation and the like. The methodfor transfer can be selected so as to provide for stable expression ofthe introduced polypeptide-encoding nucleic acid. Thepolypeptide-encoding nucleic acid can be provided as an inheritableepisomal element (e.g., plasmid) or can be genomically integrated.

Viral vectors can also be used for cloning and expression of the nucleicacids disclosed herein. Exemplary plant viral vectors includecauliflower mosaic virus (CaMV), pea early browning virus (PEBV), beanpod mottle virus (BPMV), cucumber mosaic virus (CMV), apple latentspherical virus (ALSV), tobacco mosaic virus (TMV), potato virus X,brome mosaic virus (BMV) and barley stripe mosaic virus (BSMV).

Additional vectors can be used for expression of C. subtsugaepolypeptide sequences in non-plant organisms. These include prokaryoticcloning vectors (e.g., pBR322, pUC, bacteriophage lambda), fungalvectors (e.g., yeast 2-micron plasmid), insect cloning vectors (e.g.,baculovirus) and mammalian vectors (e.g., SV40).

Suitable vectors for transferring a polypeptide-encoding nucleic acidcan vary in composition. Integrative vectors can be conditionallyreplicative or suicide plasmids, bacteriophages, and the like. Theconstructs can include various elements, including for example,promoters, selectable genetic markers (e.g., genes conferring resistanceto antibiotics, for example, instance neomycin, G418, methotrexate,ampicillin kanamycin, erythromycin, chloramphenicol, or gentamycin),origins of replication (to promote replication in a host cell, e.g., abacterial host cell), and the like. The choice of vector depends upon avariety of factors such as the type of cell in which propagation isdesired and the purpose of propagation. Certain vectors are useful foramplifying and making large amounts of the desired DNA sequence. Othervectors are suitable for expression of protein in cells. Still othervectors are suitable for transfer and expression in cells in a wholeanimal or plant. The choice of appropriate vector is well within theskill of the art. Many such vectors are available commercially.

The vector used can be an expression vector based on episomal plasmidscontaining selectable drug resistance markers and elements that providefor autonomous replication in different host cells. Vectors are amplydescribed in numerous publications well known to those in the art,including, e.g., Short Protocols in Molecular Biology, (1999) F.Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expressionof the nucleic acids encoding the subject polypeptide, may provide forpropagating the subject nucleic acids, or both.

Constructs can be prepared by, for example, inserting a polynucleotideof interest into a construct backbone, typically by means of DNA ligaseattachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination or site-specific recombination, or by one ormore amplification methods (e.g., PCR). Typically homologousrecombination is accomplished by attaching regions of homology to thevector on the flanks of the desired nucleotide sequence, whilesite-specific recombination can be accomplished through use of sequencesthat facilitate site-specific recombination (e.g., cre-lox, att sites,etc.). Nucleic acid containing such sequences can be added by, forexample, ligation of oligonucleotides, or by polymerase chain reactionusing primers comprising both the region of homology and a portion ofthe desired nucleotide sequence.

For expression of the polypeptide of interest, an expression cassettecan be employed. Thus, the present disclosure provides a recombinantexpression vector comprising a subject nucleic acid. The expressionvector can provide transcriptional and translational regulatorysequences, and can also provide for inducible or constitutiveexpression, wherein the coding region is operably placed under thetranscriptional control of a transcriptional initiation region (e.g., apromoter, enhancer), and transcriptional and translational terminationregions. These control regions may be native to the C. subtsugae genome,or can be derived from exogenous sources. As such, control regions fromexogenous sources can be considered heterologous elements that areoperably linked to the nucleic acid encoding the subject polypeptide. Ingeneral, the transcriptional and translational regulatory sequences caninclude, but are not limited to, promoter sequences, operator sequences,ribosomal binding sites, transcriptional start and stop sequences,translational start and stop sequences, polyadenylation sites andenhancer or activator sequences. Promoters can be either constitutive orinducible, and can be a strong constitutive promoter (e.g., T7 promoter,SP6 promoter, and the like).

Exemplary plant regulatory sequences, which can be used in therecombinant constructs disclosed herein, include constitutive promoterssuch as the CaMV 19S and 35S promoters and those from genes encodingactin or ubiquitin. Alternatively, regulated promoters such aschemically-regulated promoters (e.g., tetracycline-regulated) andwound-inducible promoters (expressed at wound sites and at sites ofphytopathogenic infection) can also be used. In additional embodiments,promoters can be tissue-specific (e.g., specifying expression in roots,leaves, flowers, inflorescences) and/or temporally regulated (e.g.,specifying expression in seedlings).

Additional promoters for use in plant cells have been described. See,for example, Stanford et al. (1989) Mol. Gen. Genet. 215: 200-208; Xu etal. (1993) Plant Molec. Biol. 22: 573-588; Logemann et al. (1989) PlantCell 1: 151-158; Rohrmeier & Lehle (1993) Plant Molec. Biol. 22:783-792; Firek et al. (1993) Plant Molec. Biol. 22: 129-142 and Warneret al. (1993) Plant J. 3: 191-201.

Consensus plant translation initiation sequences (i.e., ribosome-bindingsites) have been described by Joshi (1987) Nucleic Acids Res.15:6643-6653 and in the Clontech Catalogue 1993/1994, page 210.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding proteins of interest. A selectable marker operativein the expression host can be present to facilitate selection of cellscontaining the vector. In addition, the expression construct can includeadditional elements. For example, the expression vector can have one ortwo replication systems, thus allowing it to be maintained, for example,in plant or insect cells for expression and in a prokaryotic host forcloning and amplification. In addition, the expression construct cancontain a selectable marker gene to allow the selection of transformedhost cells. Selection genes are known in the art and vary depending onthe host cell used.

Expression vectors provided herein contain the aforementioned nucleicacids and/or polynucleotides. Such expression vectors can containpromoters (e.g., T7 promoter, T3 promoter, SP6 promoter, E. coli RNApolymerase promoter, lac promoter and its derivatives, tac promoter, trppromoter, the arabinose-inducible P_(BAD) promoter, theL-rhamnose-inducible rhaP_(BAD) promoter, bacteriophage lambda promoters(e.g, P_(L)), CMV promoter, SV40 promoter, PGK promoter, EF-1alphapromoter), operators, transcription termination signals (e.g., SV40termination signal), splice sites (e.g., SV40 splice sites, beta-globinsplice site), ribosome binding sites, signal sequences (e.g.,immunoglobulin kappa signal sequence), epitopes tags (e.g., myc, FLAG),purification tags (e.g., His₆), replication origins and drug selectionmarkers. Linker sequences, encoding linker amino acids and/or comprisingrestriction enzyme recognition sites, or any other type of linkersequence, can also be operably linked to the nucleic acid encoding thesubject polypeptide present in the vectors disclosed herein.

Cosmid libraries can be prepared by methods known in the art. See, forexample, Maniatis et al. Molecular Cloning: A Laboratory Manual. ColdSpring Harbor Press, 2^(nd) edition, 1989 and Sambrook et al., 2001.Such a library can be used for sequence-based screening and for any typefunctional screening of cells, or of supernatants, whole cell broths,cell-free lysates, or extracts derived from the cells. High throughputbiological assays for herbicidal screening, enzymatic activities,anti-cancer activity, etc. are known in the art and described in theliterature. See also Examples 7-11 herein.

Host Cells

The present disclosure further contemplates recombinant host cellscontaining an exogenous polynucleotide. Said polynucleotide can compriseone or more fragments of the C. subtsugae genes as disclosed herein, orcan encode one or more of the polypeptides of the present disclosure.Host cells can be procaryotic (e.g., bacterial) or eucaryotic (e.g.,yeast, insect, mammalian). The host can also be a synthetic cell.

In certain embodiments, the host cell is a microorganism. Suitablemicroorganisms are those capable of colonizing plant tissue (e.g. root,stems, leaves, flowers, internally and on the surface), or therhizosphere, in such manner that they come in contact with insect pests.Some of the host microorganisms can also be capable of colonizing thegut of an insect pest, and be capable of being transmitted from oneinsect to another. Host microorganisms can also colonize the gut andbody surface of a plant pest. The host cell can also be used as amicrobial factory for the production of C. subtsugae proteins, or forproduction of one or more compounds produced by the activity of C.subtsugae proteins such as, for example, peptides, lipids, lipopeptides,glycoproteins, secondary metabolites, antibiotics and small organiccompounds.

Gram-negative microorganisms suitable for heterologous expressioninclude: Escherichia coli (e.g., E. coli K12, E. coli BL21), Pseudomonassp. (e.g. Pseudomonas fluorescens, Pseudomonas putida, Psuedomonasaurantiaca, Psuedomonas aureofaciens, Psuedomonas protegens),Enterobacter sp. (e.g. Enterobacter cloacae), and Serratia sp. ExemplaryE. coli strains include E. coli BL21 and E. coli K12 for routineexpression. Other E. coli strains, for more specialized purposes, arethose which display protease deficiency (BL21-B838) and those whichoverexpress membrane proteins such as the BL21 derivative DE3, C41 (DE3)and C43 (DE3).

Methods for high-level expression of heterologous proteins in E. coliare known and include (a) IPTG-induction methods, (b) auto-inductionmethods, and (c) high cell-density IPTG-induction methods. See, forexample, Sivashanmugam et al. (2009).

Gram-positive microorganisms suitable for heterologous expressioninclude Bacillus sp. (e.g., Bacillus megaterium, Bacillus subtilis,Bacillus cereus), and Streptomyces sp. One advantage of using Bacillusas an expression host is that members of this genus produce spores,which provide formulations with better stability and longer shelf life.Expression systems based on Bacillus megaterium and Bacillus subtilisare commercially available from MoBiTec (Germany). Nucleotide sequencesof interest can be expressed in Bacillus megaterium using under thecontrol of the promoter of the xylose operon.

Fungal microorganisms suitable for heterologous expression includeTrichoderma sp., Gliocadium, Saccharomyces cerevisiae, and Pichiapastoris. Heterologous DNA can be introduced into filamentous fungi byprotoplast-mediated transformation using polyethylene glycol (PEG) or byelectroporation-based methods. Particle bombardment is another methodthat has been successfully used to transform fungal cells.

Methods and compositions for transformation of Saccharomyces cerevisiaeare known in the art. For example, a nucleic acid can be cloned into asuitable vector (e.g., the YES vectors (Invitrogen, Carlsbad, Calif.),under the control of an inducible promoter such as GAL1, and the CYC1terminator, and expressed in Saccharomyces cerevisiae. The resultingcells can be tested for the desired activity, or for protein expression.

Heterologous expression can also be conducted in other yeast species(Jeffries et al., 2010), such as Pichia pastoris, Hansenula polymorpha,Arxula adenivorans and Yarrowia lipolytica. Transformation of Pichiapastoris can be achieved with the use of a commercial kit, such as thePichiaPink Expression System (Invitrogen, Carlsbad, Calif.), the PichiaClassic Protein Expression System or the Pichia GlycoSwitch (forglycosylated proteins) (Research Corporation Technologies, Tucson,Ariz.). For transformation of the yeasts Pichia pastoris or Hansenula.polymorpha, electroporation can also be used.

In certain embodiments, non-pathogenic symbiotic bacteria, which areable to live and replicate within plant tissues (i.e., endophytes), ornon-pathogenic symbiotic bacteria, which are capable of colonizing thephyllosphere or the rhizosphere (i.e., epiphytes) are used. Suchbacteria include bacteria of the genera Agrobacterium, Alcaligenes,Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia,Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomycesand Xanthomonas.

Symbiotic fungi, such as Trichoderma and Gliocladium can also be used ashosts for propagation and/or expression of the sequences disclosedherein.

Formulations and Pesticidal Compositions

The present disclosure provides pesticidal (e.g., insecticidal)compositions and formulations comprising the nucleic acids andpolypeptides disclosed herein.

A “pest” is an organism (prokaryotic, eucaryotic or Archael) thatincreases mortality and/or slows, stunts or otherwise alters the growthof a plant. Pests include, but are not limited to, nematodes, insects,fungi, bacteria, and viruses.

A “pesticide” as defined herein, is a substance derived from abiological product, or a chemical substance, that increases mortalityand/or inhibits the growth rate of plant pests. Pesticides include butare not limited to nematocides, insecticides, herbicides, plantfungicides, plant bactericides, and plant viricides.

A “biological pesticide” as defined herein is a microorganism withpesticidal properties.

A “pesticidal composition” is a formulation comprising a pesticide andoptionally one or more additional components. Additional componentsinclude, but are not limited to, solvents (e.g., amyl acetate, carbontetrachloride, ethylene dichloride; kerosene, xylene, pine oil, andothers listed in EPA list 4a and 4b etc.), carriers, (e.g., organicflour, Walnut shell flour, wood bark), pulverized mineral (sulfur,diatomite, tripolite, lime, gypsum talc, pyrophyllite), clay(attapulgite bentonites, kaolins, volcanic ash, and others listed in EPAlist 4a and 4b), stabilizers, emulsifiers (e.g., alkaline soaps, organicamines, sulfates of long chain alcohols and materials such as alginates,carbohydrates, gums, lipids and proteins, and others listed in EPA list4a and 4b), surfactants (e.g., those listed in EPA list 4a and 4b),anti-oxidants, sun screens, a second pesticide, either chemical orbiological (e.g., insecticide, nematicide, miticide, algaecide,fungicide, bactericide), an herbicide an/or an antibiotic.

A “carrier” as defined herein is an inert, organic or inorganicmaterial, with which the active ingredient is mixed or formulated tofacilitate its application to plant or other object to be treated, orits storage, transport and/or handling.

Pesticidal compositions as disclosed herein are useful for modulatingpest infestation in a plant. The term “modulate” as defined herein isused to mean to alter the amount of pest infestation or rate of spreadof pest infestation. Generally, such alteration is a lowering of thedegree and/or rate and/or spread of the infestation.

The term “pest infestation” as defined herein, is the presence of a pestin an amount that causes a harmful effect including a disease orinfection in a host population or emergence of an undesired weed in agrowth system. Exemplary plant pests include, but are not limited to,mites (e.g., Tetranychus urticae (Two-spotted spider mite)), fruit flies(e.g., Drosophila suzukii, Drosophila melanogaster), house flies (e.g.,Musca domestica), arachnids (e.g., Acari spp.), root maggots(Anthomyidae spp., e.g. Cabbage Root Maggots), aphids (e.g., Myzuspersicae (green peach aphid)), Triozidae spp. (e.g., potato psyllid(Bactericera cockerelli)), beetles (Tenebrionidae spp., e.g., litterbeetles (Alphitobius diaperinus)), grubs (e.g., white grub (Cyclocephalalurida), Southern Masked Chafer (Rhizotrogus majalis), Japanese beetle(Popillia japonica) larvae, black vine weevil (Otiorhyncus sulcatus)larvae, Oriental beetle (Anomala orientalis) larvae, scarabs (e.g.,Scarabaeidae spp.), nematodes (e.g., Root-knot nematode (Meloidogynespp.)), fungi, bacteria, and various plant viruses, for example, Tobaccomosaic virus, Tomato spotted wilt virus, Tomato yellow leaf curl virus,Cucumber mosaic virus, Potato virus Y, Cauliflower mosaic virus, Africancassava mosaic virus, Plum pox virus, Brome mosaic virus, Potato virusX, Citrus tristeza virus, Barley yellow dwarf virus, Potato leaf rollvirus and Tomato bushy stunt virus.

Pesticidal compositions, as disclosed herein, can be used either forprophylactic or modulatory purposes. When provided prophylactically, thecompositions(s) are provided in advance of any symptoms of infestation.The prophylactic administration of the composition(s) serves to prevent,attenuate, or decrease the rate of onset of any subsequent infection orinfestation. When provided for modulatory purposes, the composition(s)are provided at (or shortly after) the onset of an indication ofinfection or infestation. Modulatory administration of the compound(s)serves to attenuate the pathological symptoms of the infection orinfestation and to increase the rate of recovery.

Additional methods can be employed to control the duration of action.Controlled-release can be achieved through the use of polymers tocomplex or absorb one or more of the components of the composition. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate compositionsas disclosed herein into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. Alternatively, instead of incorporating thesecompositions into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldelivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are known in the art.

Pesticidal compositions as disclosed herein, (e.g., pesticidal toxins)can be produced by expression of selected Chromobacterium subtsugae genesequences in heterologous hosts suitable for lab scale, pilot scale andmanufacturing scale fermentation (e.g., E. coli, Psuedomonas sp., yeast,etc.). Toxins can be produced by fermentation procedures known in theart using the heterologous host and formulated directly, or afterextraction and purification of the toxin from the fermentation broth.The formulation can include live cells or non-viable cells.

The pesticidal compositions disclosed herein can be formulated in anymanner. Non-limiting formulation examples include, but are not limitedto, emulsifiable concentrates (EC), wettable powders (WP), solubleliquids (SL), aerosols, ultra-low volume concentrate solutions (ULV),soluble powders (SP), microencapsulates, water dispersed granules,flowables (FL), microemulsions (ME), nano-emulsions (NE), etc. In any ofthe formulations described herein, the percentage of the activeingredient is within a range of 0.01% to 99.99%. Detailed description ofpesticide formulations can be found in the Kirk-Othmer Encyclopedia ofChemical Technology; Knowles, A. 2005. New Developments in CropProtection Product Formulation, Agrow Reports, London, UK; Valkenburg,W. van (ed.) 1973, Pesticide Formulation, Marcel Dekker, New York, USA;Knowles, D. A. (ed.) 1998, Chemistry and Technology of AgrochemicalFormulations, Kluwer Academic Publishers, Dordrecht, the Netherlands.

Powder and Dust Formulations

These are simple formulations that usually contain 0.1-25% of the activeingredient. However, higher concentrations of active ingredient can beused depending on the potency and particular application. The pesticidetoxin is mixed with a solid carrier, preferably of small particle size.Solid carriers can include: silicate clays (e.g., attapulgite,bentonites, volcanic ash, montmorillionite, kaolin, talc, diatomites,etc.), carbonates (e.g., calcite, dolomite, etc), synthetics(precipitated silica, fumed silica, etc.), ground botanicals (e.g., corncob grits, rice hulls, coconut shells, etc.), organic flour (e.g.,Walnut shell flour, wood bark, etc.) or pulverized mineral (e.g.,Sulphur, diatomite, tripolite, lime, gypsum talc, pyrophyllite, etc.).The inert ingredients used in dust formulations can also come from thoselisted in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations. Small particle size can be achieved by mixingthe active ingredient with the carrier and pulverizing in a mill. Dustsare defined as having a particle size less than 100 microns; and withincrease in particle size the toxicity of the formulation decreases. Inthe selection of a dust formulation its compatibility, fineness, bulkdensity, flow ability, abrasiveness, absorbability, specific gravity andcost should be taken into consideration. Exemplary dust formulations areprovided in Table 1.

TABLE 1 Formulation Formu- Formu- components Formulation A Formulation Blation C lation D Active ingredient 0.65 5 10 25 Talc 50 90 Kaolin orother clay 49.35 95 75

A dust formulation can also be prepared from a dust concentrate (e.g.,40% active ingredient, 5% stabilizer, 20% silica, 35% magnesiumcarbonate) added at 1-10% to a 1:1 organic filler/talc combination.

The dust formulation is used as a contact powder (CP) or tracking powder(TP) against crawling insects.

A dust formulation with high flowability can be applied by pneumaticequipments in greenhouses.

Granular and Pellet Formulations

The pesticidal toxin is applied in liquid form to coarse particles ofporous material (e.g., clay, walnut shells, vermiculite, diatomaceousearth, corn cobs, attapulgite, montmorillioinite, kaolin, talc,diatomites, calcite, dolomite, silicas, rice hulls, coconut shells,etc.). The granules or pellets can be water dispersible, and can beformed by extrusion (for pesticidal actives with low water solubility),agglomeration or spray drying. Granules can also be coated orimpregnated with a solvent-based solution of the pesticidal toxin. Thecarrier particles can be selected from those listed in EPA Inert List 4a(www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) for conventionalformulations and 4b (www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf)for organic formulations. The active ingredient can be absorbed by thecarrier material or coated on the surface of the granule. Particle sizecan vary from 250 to 1250 microns (0.25 mm to 2.38 mm) in diameter. Theformulations usually contain 2 to 10 percent concentration of thetoxicant. The granules are applied in water or whorls of plant or tosoil at the rate of 10 kg/ha. Granular formulations of systemicinsecticides are used for the control of sucking and soil pest byapplication to soil. Whorl application is done for the control of borerpests of crops such as sorghum, maize and sugarcane, etc. These types offormulations reduce drift and allow for slower release of the pesticidalcomposition.

Granular pesticides are most often used to apply chemicals to the soilto control weeds, fire ants, nematodes, and insects living in the soilor for absorption into plants through the roots. Granular formulationsare sometimes applied by airplane or helicopter to minimize drift or topenetrate dense vegetation. Once applied, granules release the activeingredient slowly. Some granules require soil moisture to release theactive ingredient. Granular formulations also are used to control larvalmosquitoes and other aquatic pests. Granules are used in agricultural,structural, ornamental, turf, aquatic, right-of-way, and public health(biting insect) pest control operations.

Application of granular formulations is common in pre-emergenceherbicides or as soil insecticides for direct application andincorporation into soil or other solid substrates where plants grow.Granules or pellets can also be applied in-furrow. Granules are commonlyused for application to water, such as in flooded rice paddies.

A typical granule formulation includes (% w/w) 1-40% active ingredient,1-2% stabilizer, 0-10% resin or polymer, 0-5% surfactant, 0-5% binderand is made up to 100% with the carrier material.

Wettable Powder Formulations

Wettable powder is a powdered formulation which yields a rather stablesuspension when diluted with water. It is formulated by blending thepesticidal agent with diluents such as attapulgite, a surface activeagent and auxiliary materials such as sodium salts of sulfo acids.Optionally stickers are added to improve retention on plants and othersurfaces. Wettable powders can be prepared by mixing the pesticidaltoxin (10-95%) with a solid carrier, plus 1-2% of a surface-active agentto improve suspensibility. The overall composition of the formulationincludes the active ingredient in solid form (5.0-75%), an anionicdispersant and an anionic or nonionic wetting agent.

A typical example of a wettable powder formulation includes 10-80%active ingredient, 1-2% wetting agents (e.g., benzene sulphonates,naphthalene sulphonates, aliphatic suplhosuccinates, aliphatic alcoholetoxylates, etc.), 2-5% dispersing agent (e.g., lignosulphonates,naphthalene sulphonate-formaldehyde condensates, etc.), and 0.1-1%antifoaming agent (e.g., isopar M (Exxon/Mobil)), made up to 100% withan inert filler or carrier (e.g., diatomaceous earth, silica, etc.).

Emulsifiable Concentrate (EC) Formulations

These are concentrated pesticide formulation containing an organicsolvent and a surfice-active agent to facilitate emulsification withwater. When EC formulations are sprayed on plant parts, the solventevaporates quickly, leaving a deposit of toxin from which water alsoevaporates. Exemplary emulsifying agents in insecticide formulationsinclude alkaline soaps, organic amines, sulfates of long chain alcoholsand materials such as alginates, carbohydrates, gums, lipids andproteins. Emulsifying agents can be selected from those listed in EPAInert List 4a (www.epa.gov/opprd001/inerts/inerts_list4Acas.pdf) forconventional formulations and 4b(www.epa.gov/opprd001/inerts/inerts_list4Bname.pdf) for organicformulations.

Solution Formulations

A solution formulation is a concentrated liquid pesticide formulationthat can be used directly, or require dilution in the case of a solubleconcentrate. Soluble concentrates and solutions are water- orsolvent-based mixtures with complete miscibility in water.

A typical example of a solution concentrate formulation includes 20-70%active ingredient, 5-15% wetting agent, 5-10% antifreeze, and is made upto 100% with water or a water miscible solvent.

Depending on the nature and stability of the pesticidal toxin, asolution formulation can optionally include thickeners, preservatives,antifoam, pH buffers, UV screens, etc.

Aerosol and Fumigant Formulations

In an insecticidal aerosol, the toxin is suspended as minute particleshaving sizes ranging from 0.1 to 50 microns in air as a fog or mist.This is achieved by burning the toxin or vaporizing it by heating. Thetoxicant dissolved in a liquefied gas, if released through small hole,may cause the toxicant particles to float in air with the rapidevaporation of the released gas.

A chemical compound, which is volatile at ambient temperatures andsufficiently toxic, is known as a fumigant. Fumigants generally enter aninsect via its tracheal system. Fumigants are used for the control ofinsect pests in storage bins, buildings and certain insects andnematodes in the soil. Most fumigants are liquids held in cans or tanksand often comprise mixtures of two or more gases. Alternatively,phosphine or hydrogen phosphide gas can be generated in the presence ofmoisture from a tablet made up of aluminium phosphide and ammoniumcarbonate. The advantage of using a fumigant is that sites that are noteasily accessible to other chemicals can be reached with fumigants, dueto the penetration and dispersal of the gas. Commonly used fumigants areEDCT, methyl bromide, aluminium phosphide and hydrocynic acid.

Formulation in Fertilizers Mixtures

A fertilizer mixture can be manufactured by addition of an insecticidalcomposition, as disclosed herein, to a chemical fertilizer, or byspreading the composition directly on the fertilizer. Fertilizermixtures are applied at the regular fertilizing time and provide bothplant nutrients and control of soil insects. In an exemplary fertilizerformulation, urea (2% solution) is mixed with an insecticidalcomposition and sprayed for supply of nitrogen to the plant and forrealizing effective pest control.

Formulation as Poison Baits.

Poison baits consist of a base or carrier material attractive to thepest species and a chemical toxicant in relatively small quantities. Thepoison baits are used for the control of fruit flies, chewing insects,wireworms, white grubs in the soil, household pests, rats in the fieldand slugs. These formulations are useful for situations in which sprayapplication is difficult. A common base used in dry baits is wheat branmoistened with water and molasses. For the control of fruit suckingmoths fermenting sugar solution or molasses with a toxin is used.

Formulations for Seed Treatments

Seed treatments include application of a pesticidal composition,optionally in combination with other bioactive, antagonistic orsymbiotic agents, to the surface of a seed prior to sowing. Thepesticidal toxins, proteins, and/or compounds disclosed herein can beformulated for seed treatments in any of the following modes: drypowder, water slurriable powder, liquid solution, flowable concentrateor emulsion, emulsion, microcapsules, gel, or water dispersiblegranules; or can be applied to seeds by spraying on the seed beforeplanting.

In the case of a dry powder, the active ingredient is formulatedsimilarly to a wettable powder, but with the addition of a stickingagent, such as mineral oil, instead of a wetting agent. For example: onekg of purified talc powder (sterilized for 12 h), 15 g calciumcarbonate, and 10 g carboxymethyl cellulose are mixed under asepticconditions following the method described by Nandakumar et al (2001).Protein, nucleic acid suspensions or organisms expressing these aremixed in a 1:2.5 ratio (suspension to dry mix) and the product is shadedried to reduce moisture content to 20-35%.

The compositions can be in the form of a liquid, gel or solid.

A solid composition can be prepared by suspending a solid carrier in asolution of active ingredient(s) and drying the suspension under mildconditions, such as evaporation at room temperature or vacuumevaporation at 65° C. or lower. For liquid compositions, the activeingredient can be dissolved in a suitable carrier or solvent.

A composition can comprise gel-encapsulated active ingredient(s). Suchgel-encapsulated materials can be prepared by mixing a gel-forming agent(e.g., gelatin, cellulose, or lignin) with a composition comprising oneor more nucleic acids and/or polypeptides as disclosed herein, andoptionally a second pesticide or herbicide; and inducing gel formationof the agent.

The composition can additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)monolaurate. The concentration of surfactants can range between 0.1-35%of the total formulation, e.g., from 5-25%. The choice of dispersing andemulsifying agents, such as non-ionic, anionic, amphoteric and cationicdispersing and emulsifying agents, and the amount employed, isdetermined by the nature of the composition and the ability of the agentto facilitate the dispersion of the composition.

Formulations Comprising Microorganisms

Pesticidal compositions as set forth above can be combined with amicroorganism. The microorganism can be a plant growth promoter.Suitable microorganisms include, but are not limited to, Bacillus sp.(e.g., Bacillus firmus, Bacillus thuringiensis, Bacillus pumilus,Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis),Paecilomyces sp. (P. lilacinus), Pasteuria sp. (P. penetrans),Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., Ampelomyces sp.,Pseudozyma sp., Streptomyces sp (S. bikiniensis, S. costaricanus, S.avermitilis), Burkholderia sp., Trichoderma sp., Gliocladium sp.,avermectin, Myrothecium sp., Paecilomyces spp., Sphingobacterium sp.,Arthrobotrys sp., Chlorosplenium sp., Neobulgaria sp., Daldinia sp.,Aspergillus sp., Chaetomium sp., Lysobacter sp., Lachnum papyraceum,Verticillium suchlasporium, Arthrobotrys oligospora, Verticilliumchlamydosporium, Hirsutella rhossiliensis, Pochonia chlamydosporia,Pleurotus ostreatus, Omphalotus olearius, Lampteromyces japonicas,Brevudimonas sp., Muscodor sp., Photorhabdus sp., and Burkholderia sp.Agents obtained or derived from such microorganisms can also be used incombination with the pesticidal nucleic acids and polypeptides disclosedherein.

Formulations Comprising Second Pesticides

Pesticidal compositions as set forth above can be combined with a secondpesticide (e.g., nematocide, fungicide, insecticide, algaecide,miticide, or bactericide). Such an agent can be a natural oil oroil-product having fungicidal, bactericidal, nematicidal, acaricidaland/or insecticidal activity (e.g., paraffinic oil, tea tree oil,lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil,pyrethram). Furthermore, the pesticide can be a single site anti-fungalagent which may include but is not limited to benzimidazole, ademethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine,triazole), morpholine, hydroxypyrimidine, anilinopyrimidine,phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide,carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatichydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin,acylamine, phthalimide, benzenoid (xylylalanine); a demethylationinhibitor selected from the group consisting of imidazole, piperazine,pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole,propiconazole, triadimefon, bromuconazole, cyproconazole, diniconazole,fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil,an anthranilic diamide (e.g., chlorantranilipole) and a quinone outsideinhibitor (e.g., strobilurin). The strobilurin may include but is notlimited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). Theanti-fungal agent can also be derived from a Reynoutria extract.

The fungicide can also be a multi-site non-inorganic, chemical fungicideselected from the group consisting of chloronitrile, quinoxaline,sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios,phenylpyridin-amine, and cyano-acetamide oxime.

The composition can, as noted above, further comprise an insecticide.The insecticide can include but is not limited to avermectin, Bt (e.g.,Bacillus thuringiensis var. kurstaki), neem oil, spinosads, Burkholderiasp. (e.g., as set forth in WO2011/106491), entomopathogenic fungi such aBeauveria bassiana and chemical insecticides including but not limitedto organochlorine compounds, organophosphorous compounds, carbamates,pyrethroids, pyrethrins and neonicotinoids.

As noted above, the composition may further comprise a nematocide. Thisnematocide may include, but is not limited to, avermectin, microbialproducts such as Biome (Bacillus firmus), Pasteuria spp and organicproducts such as saponins.

Methods for Modulating Pest Infestation

Thus, according to the present disclosure, methods for modulating pestinfestation in a plant are provided. The methods comprise application toa plant, or to the soil or substrate in which the plant is growing, of apesticidal composition comprising a nucleic acid as disclosed herein;i.e., any of SEQ ID NOs:4-6.

Additional methods for modulating pest infestation in a plant compriseapplication, to a plant, or to the soil or substrate in which the plantis growing, of a pesticidal composition comprising a polypeptide asdisclosed herein; i.e., any of SEQ ID NOs:1-3.

When used as biological insect control agents, insecticidal toxinsencoded by the C. subtsugae genome can be produced by expression of a C.subtsugae nucleotide sequence in a heterologous host cell capable ofexpressing the nucleotide sequences. In one embodiment, one or more C.subtsugae nucleotide sequences are inserted into an appropriateexpression cassette comprising, e.g., a promoter and a transcriptionaltermination signal. Expression of the nucleotide sequence(s) can beconstitutive or inducible, depending on the promoter and/or externalstimuli. In certain embodiments, the cell in which the toxin isexpressed is a microorganism, such as a virus, a bacterium, or a fungus.

In certain embodiments, a virus, such as a baculovirus, is engineered tocontain a C. subtsugae nucleotide sequence in its genome. Such arecombinant virus can express large amounts of, e.g., an insecticidaltoxin after infection of appropriate eukaryotic cells that are suitablefor virus replication and expression of the nucleotide sequence. Theinsecticidal toxin thus produced is used as an insecticidal agent.Alternatively, baculoviruses engineered to include the nucleotidesequence are used to infect insects in vivo and kill them, either byexpression of the insecticidal toxin or by a combination of viralinfection and expression of the insecticidal toxin.

Thus, the compositions set forth above, comprising C. subtsugae nucleicacids and polypeptides, can be used as pesticides. In particular, thecompositions as set forth above can be used as, for example,insecticides and nematicides, alone or in combination with one or moresecond pesticidal substances as set forth herein.

Specifically, nematodes that may be controlled using the method setforth above include but are not limited to parasitic nematodes such asroot-knot, cyst, and lesion nematodes, including but not limited to seedgall nematodes (Afrina wevelli), bentgrass nematodes (Anguina agrostis),shoot gall nematodes (Anguina spp.), seed gall nematodes (Anguina spp.,A. amsinckiae, A. balsamophila; A. tritici), fescue leaf gall nematodes(A. graminis), ear-cockle (or wheat gall) nematodes (Anguina tritici),bud and leaf (or foliar) nematodes (Aphelenchoides spp., A. subtenuis),begonia leaf (or fern, or spring crimp, or strawberry foliar, orstrawberry nematodes, or summer dwarf) nematodes (A. fragariae), fernnematodes (A. olesistus), rice nematodes (A. oryzae), currant nematodes(A. ribes), black currant (or chrysanthemum) nematodes (A. ritzemabosi),chrysanthemum foliar or leaf nematodes (A. ritzemabosi), rice white-tip(or spring dwarf, or strawberry bud) nematodes (A. besseyi),fungus-feeding (mushroom) nematodes (Aphelenchoides composticola),Atalodera spp. (Atalodera lonicerae, Atalodera ucri), spine nematodes(Bakernema variabile), sting nematodes (Belonolaimus spp., B. gracilis,B. longicaudatus), pine wood nematodes (Bursaphalenchus spp., B.xylophilus, B. mucronatus), sessile nematodes (Cacopaurus spp., C.epacris, C. pestis), amaranth cyst nematodes (Cactodera amaranthi),birch cyst nematodes (C. betulae), cactus cyst nematodes (C. cacti),estonian cyst nematodes (C. estonica), Thorne's cyst nematodes (C.thornei), knotweed cyst nematodes (C. weissi), ring nematodes (Criconemaspp.), spine nematodes (Criconema spp., C. civellae, C. decalineatum, C.spinalineatum), ring nematodes (Criconemella axeste, C. curvata, C.macrodora, C. parva), ring nematodes (Criconemoides spp., C. citri, C.simile), spine nematodes (Crossonema fimbriatum), eucalypt cystoidnematodes (Cryphodera eucalypti), bud, stem and bulb nematodes(Ditylenchus spp., D. angustus, D. dipsaci, D. destructor, D.intermedius), Mushroom spawn nematodes (D. myceliophagus), awl nematodes(Dolichodorus spp., D. heterocephalus, D. heterocephalous), spearnematodes (Dorylaimus spp.), stunt nematodes (Geocenamus superbus), cystnematodes (Globodera spp.), yarrow cyst nematodes (G. achilleae),milfoil cyst nematodes (G. millefolii), apple cyst nematodes (G. mali),white cyst potato nematodes (G. pallida), golden nematodes (G.rostochiensis), tobacco cyst nematodes (G. tabacum), Osborne's cystnematodes (G. tabacum solanacearum), horsenettle cyst nematodes (G.tabacum virginiae), pin nematodes (Gracilacus spp., G. idalimus), spiralnematodes (Helicotylenchus spp., H. africanus, H. digonicus, H.dihystera, H. erythrinae, H. multicinctus, H. paragirus, H.pseudorobustus, H. solani, H. spicaudatus), sheathoid nematodes(Hemicriconemoides spp., H. biformis, H. californianus, H. chitwoodi, H.floridensis, H. wessoni), sheath nematodes (Hemicycliophora spp., H.arenaria, H. biosphaera, H. megalodiscus, H. parvana, H. poranga, H.sheri, H. similis, H. striatula), cyst nematodes (Heterodera spp.),almond cyst nematodes (H. amygdali), oat (or cereal) cyst nematodes (H.avenae), Cajanus (or pigeon pea) cyst nematodes (H. cajani),bermudagrass (or heart-shaped, or Valentine) cyst nematodes (H.cardiolata), carrot cyst nematodes (H. carotae), cabbage cyst nematodesor brassica root eelworm (H. cruciferae), nutgrass (or sedge) cystnematodes (H. cyperi), Japanese cyst nematodes (H. elachista), fig (orficus, or rubber) cyst nematodes (H. fici), galeopsis cyst nematodes (H.galeopsidis), soybean cyst nematodes (H. glycines), alfalfa root (or peacyst) nematodes (H. goettingiana), buckwheat cyst nematodes (H.graduni), barley cyst nematodes (H. hordecalis), hop cyst nematodes (H.humuli), Mediterranean cereal (or wheat) cyst nematodes (H. latipons),lespedeza cyst nematodes (H. lespedezae), Kansas cyst nematodes (H.longicolla), cereals root eelworm or oat cyst nematodes (H. major),grass cyst nematodes (H. mani), lucerne cyst nematodes (H. medicaginis),cyperus (or motha) cyst nematodes (Heterodera mothi), rice cystnematodes (H. oryzae), Amu-Darya (or camel thorn cyst) nematodes (H.oxiana), dock cyst nematodes (H. rosii), rumex cyst nemtodes (H.rumicis), sugar beet cyst nematodes (H. schachtii), willow cystnematodes (H. salixophila), knawel cyst nematodes (H. scleranthii),sowthistle cyst nematodes (H. sonchophila), tadzhik cyst nematodes (H.tadshikistanica), turkmen cyst nematodes (H. turcomanica), clover cystnematodes (H. trifolii), nettle cyst nematodes (H. urticae), ustinovcyst nematodes (H. ustinovi), cowpea cyst nematodes (H. vigni), corncyst nematodes (H. zeae), rice root nematodes (Hirschmanniella spp., H.belli, H. caudacrena, H. gracilis, H. oryzae), lance nematodes(Hoplolaimus spp.), Columbia nematodes (H. columbus), Cobb's lancenematodes (H. galeatus), crown-headed lance nematodes (H.tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis),needle nematodes (Longidorus spp., L. africanus, L. sylphus), ringnematodes (Macroposthonia (=Mesocriconema) xenoplax), cystoid nematodes(Meloidodera spp.), pine cystoid nematodes (M. floridensis), tadzhikcystoid nematodes (M. tadshikistanica), cystoid body nematodes(Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M.conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogynespp., M. acronea, M. arenaria, M. artiellia, M. brevicauda, M.camelliae, M. carolinensis, M. chitwoodi, M. exigua, M. graminicola, M.hapla, M. hispanica, M. incognita, M. incognita acrita, M. indica, M.inornata, M. javanica, M. kikuyuensis, M. konaensis, M. mali, M.microtyla, M. naasi, M. ovalis, M. platani, M. querciana, M. sasseri, M.tadshikistanica, M. thamesi), knapweed nematodes (Mesoanguina picridis),Douglas fir nematodes (Nacobbodera chitwoodi), false root-knot nematodes(Nacobbus aberrans, N. batatiformis, N. dorsalis), sour paste nematodes(Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes(Paralongidorus microlaimus), spiral nematodes (Pararotylenchus spp.),stubby-root nematodes (Paratrichodorus allius, P. minor, P. porosus, P.renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P.bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P.holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P.similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P.brachyurus, P. coffeae, P. convallariae, P. crenatus, P. flakkensis, P.goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P.neglectus, P. penetrans, P. pratensis, P. scribneri, P. thornei, P.vulnus, P. zeae), stem gall nematodes (Pterotylenchus cecidogenus),grass cyst nematodes (Punctodera punctate), stunt nematodes(Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholusspp.), banana-root nematodes (R. similis), rice-root nematodes (R.oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchuscocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R.parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R.christiei, R. robustus), Thorne's lance nematodes (R. uniformis),Sarisodera hydrophylla, spiral nematodes (Scutellonema spp., S.blaberum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S.conicephalum), grass root-gall nematodes (Subanguina radicicola), roundcystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes(Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T.primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (orstylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T.aspericutis, T. claytoni, T. ebriensis, T. elegans, T. golden, T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T.oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulussemipenetrans), and dagger nematodes (Xiphinema spp., X. americanum, X.bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X.diversicaudatum X. index, X. insigne, X. nigeriense, X. radicicola, X.setariae, X. vulgarae, X. vuittenezi).

Phytopathogenic insects controlled by the methods set forth aboveinclude but are not limited to non-Culicidae larvae insects from theorder (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp.,Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp.,Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp.,Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp.,Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasiaspp., Cochylis spp., Coleophora spp., Crocidolomia binotalis,Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsiscastanea, Earias spp., Ephestia spp., Eucosma spp., Eupoeciliaambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedyanubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferialycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesiabotrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestrabrassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammenespp., Pandemis spp., Panolis flammea, Pectinophora gossypiella,Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella,Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp.,Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp.,Trichoplusia ni and Yponomeuta spp.; (b) Coleoptera, for example,Agriotes spp., Anthonomus spp., Atomaria linearis, Chaetocnema tibialis,Cosmopolites spp., Curculio spp., Dermestes spp., Diabrotica spp.,Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrusspp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Phlyctinusspp., Popillia spp., Psylliodes spp., Rhizopertha spp-, Scarabeidae,Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. andTrogoderma spp.; (c) Orthoptera, for example, Blatta spp., Blattellaspp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplanetaspp. and Schistocerca spp.; (d) Isoptera, for example, Reticulitermesspp.; (e) Psocoptera, for example, Liposcelis spp.; (f) Anoplura, forexample, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigusspp. and Phylloxera spp.; (g) Mallophaga, for example, Damalinea spp.and Trichodectes spp.; (h) Thysanoptera, for example, Frankliniellaspp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaciand Scirtothrips aurantii; (i) Heteroptera, for example, Cimex spp.,Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp.,Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergellasingularis, Scotinophara spp. and Tniatoma spp.; (j) Homoptera, forexample, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp.,Aphididae, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasterspp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccushesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp.,Gascardia spp., Laodelphax spp., Lecanium corni, Lepidosaphes spp.,Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp.,Paratoria spp., Pemphigus spp., Planococcus spp., Pseudaulacaspis spp.,Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotusspp., Rhopalosiphum spp., Saissetia spp., Scaphoideus spp., Schizaphisspp., Sitobion spp., Trialeurodes vaporariorum, Trioza erytreae andUnaspis citri; (k) Hymenoptera, for example, Acromyrmex, Atta spp.,Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampaspp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsisspp. and Vespa spp.; (l) Diptera, for example, Aedes spp., Antherigonasoccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp.,Chrysomyia spp., Culex spp., Cuterebra spp., Dacus spp., Drosophilamelanogaster, Fannia spp., Gastrophilus spp., Glossina spp., Hypodermaspp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp.,Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyiahyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxysspp., Tabanus spp., Tannia spp. and Tipula spp.; (m) Siphonaptera, forexample, Ceratophyllus spp. and Xenopsylla cheopis and (n) from theorder Thysanura, for example, Lepisma saccharina.

The pesticidal compositions disclosed herein may further be used forcontrolling crucifer flea beetles (Phyllotreta spp.), root maggots(Delia spp.), cabbage seedpod weevil (Ceutorhynchus spp.) and aphids inoil seed crops such as canola (rape), mustard seed, and hybrids thereof,and also rice and maize. In a particular embodiment, the insect is amember of the Spodoptera, more particularly, Spodoptera exigua, Myzuspersicae, Plutella xylostella or Euschistus sp.

Application of an effective pesticidal control amount of a pesticidalcomposition as disclosed herein is provided. Said pesticidal compositionis applied, alone or in combination with another pesticidal substance,in an effective pest control or pesticidal amount. An effective amountis defined as that quantity of pesticidal composition, alone or incombination with another pesticidal substance that is sufficient toprevent or modulate pest infestation. The effective amount and rate canbe affected by pest species present, stage of pest growth, pestpopulation density, and environmental factors such as temperature, windvelocity, rain, time of day and seasonality. The amount that will bewithin an effective range in a particular instance can be determined bylaboratory or field tests.

Methods of Application

The pesticidal compositions disclosed herein, when used in methods formodulating pest infestation, can be applied using methods known in theart. Specifically, these compositions can be applied to plants or plantparts by spraying, dipping, application to the growth substrate (e.g.,soil) around the plant, application to the root zone, dipping rootsprior to planting, application to plants as a turf or a drench, throughirrigation, or as soil granules. Plants are to be understood as meaningin the present context all plants and plant populations such as desiredand undesired wild plants or crop plants (including naturally occurringcrop plants). Crop plants can be plants obtained by conventional plantbreeding and optimization methods, by biotechnological and geneticengineering methods or by combinations of these methods, includingtransgenic plants and plant cultivars protectable or not protectable byplant breeders' rights. Plant parts are to be understood as meaning allparts and organs of plants above and below the ground, such as shoot,leaf, flower and root, examples which may be mentioned being leaves,needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots,tubers and rhizomes. The plant parts also include harvested material,and vegetative and generative propagation material, for examplecuttings, tubers, rhizomes, off-shoots and seeds.

Application can be external, (e.g. by spraying, fogging or painting) orinternal (e.g., by injection, transfection or the use of an insectvector). When applied internally, the compositions can be intracellularor extracellular (e.g., present in the vascular system of the plant,present in the extracellular space).

Treatment of the plants and plant parts with the compositions set forthabove can be carried out directly or by allowing the compositions to acton a plant's surroundings, habitat or storage space by, for example,immersion, spraying, evaporation, fogging, scattering, painting on,injecting. In the case in which the composition is applied to a seed,the composition can be applied to the seed as one or more coats prior toplanting the seed using methods known in the art.

Pesticidal compositions as disclosed herein can also be applied toseeds; e.g., as a seed coating. Different adherents (“stickers”) can beused in the manufacture of seed coatings, including, for example, methylcellulose, alginate, carrageenan and polyvinyl alcohol. The adherent isdissolved in water to a percentage between 1-10% and stored at roomtemperature before application to the seeds. Seeds are soaked inadherent solution (3 ml/100 seeds) for 15 min, scooped out and mixedwith organic matter (1.5 g/100 seeds) in plastic bags and shakenvigorously. This process can also be automated using a seed coatingmachine.

For priming seeds with compositions as disclosed herein, seeds aresoaked in twice the seed volume of sterile distilled water containingbacterial/protein/nucleic acid suspensions or talc formulation (dryformulation) (4-10 g kg⁻¹ of seed, depending on seed size) and incubatedat 25±2° C. for 12-24 h. The suspension is then drained off and theseeds are dried under shade for 30 min and used for sowing.

The compositions can also be used as soil amendments, e.g., incombination with a carrier such as a talc formulation. Formulations forsoil amendment can also include clays, emulsifiers, surfactants andstabilizers, as are known in the art. For preparation of talc basedformulations, one kg of purified talc powder (sterilized for 12 h), 15 gcalcium carbonate, and 10 g carboxymethyl cellulose are mixed underaseptic conditions following the method described by Nandakumar et al.(2001). Protein, nucleic acid suspensions or organisms expressing theseare mixed in a 1:2.5 ratio (suspension to dry mix) and the product isshade-dried to reduce moisture content to 20-35%.

For soil amendment, formulations (e.g., talc formulations) can beapplied at rates between 2.5-10 Kg ha⁻¹ at sowing and/or at differenttimes after emergence, or both, depending on the crops.

The compositions disclosed herein can also be applied to soil usingmethods known in the art. See, for example, the USDA website atnaldc.nal.usda.gov/download/43874/pdf, accessed Feb. 20, 2013. Suchmethods include but are not limited to fumigation, drip irrigation orchemigation, broadcast application of granules or sprays, soilincorporation (e.g., application of granules), soil drenching, seedtreatment and dressing, and bare root dip.

Plant Transformation

The nucleic acids disclosed herein can be introduced into, andoptionally expressed in, plants, using any of a number of planttransformation techniques. Transformation of plants can be undertakenwith a single DNA species or multiple DNA species (i.e.,co-transformation).

In certain embodiments, a C. subtsugae protein or polypeptide (e.g., atoxin) is expressed in a plant and provides protection to the plant frominsect pests. For example, a nucleotide sequence as disclosed herein canbe inserted into an expression cassette, which can optionally be stablyintegrated into the chromosome of a plant. In certain embodiments, thenucleotide sequence is included in a non-pathogenic self-replicatingvirus. Plants transformed in accordance with the present disclosure canbe monocots or dicots and include but are not limited to, maize, wheat,barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear,quince, melon, plum, cherry, apricot, strawberry, papaya, avocado,mango, banana, alfalfa, rice, potato, eggplant, peach, cotton, carrot,tobacco, sorghum, nectarine, sugar beet, sugarcane, sunflower, soybean,tomato, pineapple, grape, raspberry, blackberry, cucumber, Arabidopsis,and woody plants such as coniferous and deciduous trees.

Once the desired nucleotide sequence has been introduced into aparticular plant species, it can be propagated in that species, ortransferred to other varieties of the same species, particularlyincluding commercial varieties, using traditional breeding techniques.

DNA can be introduced into plant cells through the use of a number ofart-recognized methods. Those skilled in the art will appreciate thatthe choice of methods can depend on the type of plant targeted fortransformation. Suitable methods for transforming plant cells are asfollows.

Agrobacterium-Mediated Transformation

A major method of DNA transfer in plants is Agrobacterium mediatedtransformation. The natural living soil bacterium Agrobacteriumtumefaciens is capable of infecting a wide range of plant species,causing Crown Gall diseases. When A. tumefaciens infects a cell, ittransfers a copy of its T-DNA, which is a small section of DNA carriedon its Ti (Tumor Inducing) plasmid. The T-DNA is flanked by two(imperfect) 25 base pair repeats. Any DNA contained within these borderswill be transferred to the host cell. Zupan and Zambriski, 1995. TheT-DNA section on the Ti plasmid can be replaced by a transgene attachedto an appropriate regulatory sequence(s). Recombinant A. tumeficienscontaining a Ti plasmid comprising exogenous nucleotide sequences canthen be used to infect cultures of either regenerating cell orprotoplasts (i.e., wall-less spherical plant cells). Marker genes suchas those coding for antibiotic resistance can be included in the Tiplasmid construct, so that it is possible to select cells that have beentransformed by the bacterium. Cell-to-plant regeneration is carried outon the selected cells by standard methods. See, for example, Zupan andZambriski (1995) and Jones et al. (2005) Plant Methods.

Agrobacterium tumefaciens can used to transform many dicotyledonousplant species with relative ease. Hinchee et al., Biotechnology6:915-921 (1988). See also Ishida et al., Nature Biotechnology14:745-750 (June 1996) for a description of maize transformation.

Biolistic Delivery

This method, also known as “particle bombardment,” involves directly“shooting” a DNA molecule into the recipient plant tissue, using a “genegun.” Tungsten or gold beads (which are smaller than the plant cellsthemselves) are coated with the DNA of interest and fired through astopping screen, accelerated by Helium, into the plant tissue. Theparticles pass through the plant cells, leaving the DNA inside. Thismethod can be used on both monocotyledonous and dicotyledonous speciessuccessfully. Transformed tissue can be selected using marker genes suchas those encoding antibiotic resistance. Whole plants, containing a copyof the transgene in all cells, can be regenerated from the totipotenttransformed cells in culture (Nottingham, 1998), using devices availablefrom Agracetus, Inc. (Madison, Wis.) and Dupont, Inc. (Wilmington,Del.).

Methods for biolistic plant transformation are known in the art. See,for example, Sanford et al., U.S. Pat. No. 4,945,050; McCabe et al.,Biotechnology 6.923-926 (1988); Weissinger et al., Annual Rev Genet.22-421-477 (1988); Sanford et al., Particulate Science and Technology5.27-37 (1987)(onion); Svab et al., Proc. Natl. Acad. Sci. USA87-8526-8530 (1990) (tobacco chloroplast); Christou et al., PlantPhysiol 87, 671-674 (1988)(soybean); McCabe et al., BioTechnology6.923-926 (1988)(soybean); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305-4309 (1988)(maize); Klein et al., BioTechnology 6, 559-563(1988) (maize); Klein et al., Plant Physiol. 91, 440-444 (1988) (maize);Fromm et al., BioTechnology 8:833-839 (1990); Gordon-Kamm et al., PlantCell 2: 603-618 (1990) (maize); Koziel et al., Biotechnology 11: 194-200(1993) (maize); Shimamoto et al., Nature 338: 274-277 (1989) (rice);Christou et al., Biotechnology 9: 957-962 (1991) (rice); Datta et al.,BioTechnology 8.736-740 (1990) (rice); European Patent Application EP 0332 581 (orchardgrass and other Pooideae); Vasil et al., Biotechnology11: 1553-1558 (1993) (wheat); Weeks et al., Plant Physiol. 102:1077-1084(1993) (wheat); Wan et al., Plant Physiol. 104:37-48 (1994) (barley);Jahne et al., Theor. Appl. Genet. 89:525-533 (1994)(barley); Umbeck etal., BioTechnology 5:263-266 (1987) (cotton); Casas et al., Proc. Natl.Acad. Sci. USA 90:11212-11216 (December 1993) (sorghum); Somers et al.,BioTechnology 10:1589-1594 (December 1992) (oat); Torbert et al., PlantCell Reports 14:635-640 (1995) (oat); Weeks et al., Plant Physiol.102:1077-1084 (1993) (wheat); Chang et al., WO 94/13822 (wheat) andNehra et al., The Plant Journal 5:285-297 (1994) (wheat).

Methods for the introduction of recombinant DNA molecules into maize bymicroprojectile bombardment can be found in Koziel et al., Biotechnology11: 194-200(1993), Hill et al., Euphytica 85:119-123 (1995) and Kozielet al., Annals of the New York Academy of Sciences 792:164-171 (1996).

Protoplast Transformation and Other Methods

Another method for the introduction of nucleic acid molecules intoplants is the protoplast transformation method for maize as disclosed inEP 0 292 435. Additional delivery systems for gene transfer in plantsinclude electroporation (Riggs et al., Proc. Natl. Acad, Sci. USA 83,5602-5606 (1986), microinjection (Crossway et al., BioTechniques 4,320-334 (1986), silicon carbide-mediated DNA transfer, direct genetransfer (Paszkowski et al., EMBO J. 3.2717-2722 (1984); Hayashimoto etal., Plant Physiol 93.857-863 (1990)(rice).

Plastid Transformation

In another embodiment, a nucleotide sequence as disclosed herein isdirectly transformed into the genome of a plastid (e.g., chloroplast).Advantages of plastid transformation include the ability of plastids toexpress bacterial genes without substantial modification of thebacterial sequences, and the ability of plastids to express multipleopen reading frames under the control of a single promoter. Plastidtransformation technology is described in U.S. Pat. Nos. 5,451,513;5,545,817 and 5,545,818; in PCT application No. WO 95/16783, and inMcBride et al. (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305.

The basic technique for chloroplast transformation involves introducingregions of cloned plastid DNA flanking a selectable marker, togetherwith the gene of interest, into a suitable target tissue using, e.g.,biolistics or protoplast transformation (e.g., calcium chloride or PEGmediated transformation). The 1 to 1.5 kb flanking regions, termedtargeting sequences, facilitate homologous recombination with theplastid genome and thus allow the replacement or modification ofspecific regions of the plastid genome. Initially, point mutations inthe chloroplast 16S rRNA and rps12 genes conferring resistance tospectinomycin and/or streptomycin were utilized as selectable markersfor transformation (Svab, Z. et al. (1990) Proc. Natl. Acad. Sci. USA87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45);resulting in the production of stable homoplasmic transformants at afrequency of approximately one per 100 bombardments of target leaves.The presence of cloning sites between these markers allowed creation ofa plastid targeting vector for introduction of foreign genes. Staub, J.M., and Maliga, P. (1993) EMBO J. 12: 601-606. Substantial increases intransformation frequency were obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial AADA gene encoding the spectinomycin-detoxifyingenzyme aminoglycoside-3′ adenyltransferase. Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90: 913-917. Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii.Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089.

Other selectable markers useful for plastid transformation are known inthe art and encompassed within the scope of the present disclosure.Typically, approximately 15-20 cell division cycles followingtransformation are required to reach a homoplastidic state. Plastidexpression, in which genes are inserted by homologous recombination intoall of the several thousand copies of the circular plastid genomepresent in each plant cell, takes advantage of the enormous copy numberadvantage, compared to nuclear genes, to achieve expression levels thatcan readily exceed 10% of the total soluble plant protein. Thus, incertain embodiments, a nucleotide sequence as disclosed herein isinserted into a plastid targeting vector and transformed into theplastid genome of a desired plant host. Plants homoplastic for plastidgenomes containing a nucleotide sequence of interest are obtained, andare capable of high-level expression of the nucleotide sequence.

Magnifection

Magnifection is a transient expression process that is based onexpression from viral RNA replicons delivered into plant cellssystemically using Agrobacterium. This method allows production ofrecombinant proteins at yields up to 5 g per kg of fresh leaf biomass,which approaches the biological limits for protein expression. Such highyields are possible because of the transient nature of the process,which allows the use of very potent amplicons derived from RNA virusessuch as Tobacco mosaic virus (TMV) or Potato virus X, without limitingbiomass accumulation, which takes place prior to infection. See, e.g.,Marillonnet et al. (2005) Nature Biotechnol. 23(6):718-723.

Additional disclosure of methods and compositions for plant geneticengineering is provided in Bircher, J A (ed.) “Plant ChromosomeEngineering: Methods and protocols.” Methods in Molecular Biology, vol.701, Springer Science+Business Media, 2011.

Transgenic Plants and Seeds

Transgenic plants derived from the plant cells can be grown to generatetransgenic plants having an enhanced trait as compared to a controlplant and produce transgenic seed and haploid pollen of this invention.Such plants with enhanced traits are identified by selection oftransformed plants or progeny seed for the enhanced trait. Forefficiency a selection method is designed to evaluate multipletransgenic plants (events) including the recombinant DNA, for examplemultiple plants from 2 to 20 or more transgenic events. Transgenicplants grown from transgenic seed provided herein demonstrate improvedagronomic traits that contribute to increased yield or other trait thatprovides increased plant value, including, for example, improved seedquality. Of particular interest are plants having enhanced water useefficiency, enhanced cold tolerance, increased yield, enhanced nitrogenuse efficiency, enhanced seed protein and enhanced seed oil. Transgenicplants of the include, but are not limited to, corn, soybean, cotton,canola, alfalfa, wheat, rice, sugarcane, sugar beet seed, millet,barley, peanut, pigeon pea, sorghum, vegetables (including but notlimited to Broccoli, Cauliflower, Cabbage, Radish, Chinese cabbage,Melons, Watermelons, Cucumber, Gourds, Pumpkin, Squash, Pepper, Tomato,Eggplant, Onion, Carrot, Garden Bean, Sweet Corn, Pea, Dry Bean, Okra,Spinach, Leek, Lettuce, and Fennel), grape, berries (including blue,black, raspberry, mullberry, boysenberry. etc), cherry and related fruittrees (including but not limited to plum, peach, apricot, kiwi,pomegranate, mango, fig), fruit trees (including but not limited toorange, lemon, lime, blood orange, grapefruit, and the like), nut trees(including but not limited to coconut, walnut (English and black),pecan, almond, hazelnut, brazil nut, hickory nut, acorn, and the like),sunflower, other oilseed producing plants or any combinations thereof.

Plant Growth Promotion

The compositions disclosed herein, in particular, C. subtsugae nucleicacids and polypeptides, can be used to modulate or more particularlypromote growth of plants, e.g. crops such as fruit (e.g., strawberry),vegetables (e.g., tomato, squash, pepper, eggplant), grain crops (e.g.,soy, wheat, rice, corn), trees, flowers, ornamental plants, shrubs(e.g., cotton, roses), bulb plants (e.g., onion, garlic) vines (e.g.,grape vine), and turf (e.g. Bermuda grass, Kentucky bluegrass, fescues).The compositions can also be used to modulate the germination of aseed(s) in a plant(s).

C. subtsugae nucleic acids and polypeptides, or a formulated productthereof, can be used alone or in combination with one or more othercomponents as described below, such as growth promoting agents and/oranti-phytopathogenic agents in a tank mix or in a program (sequentialapplication called rotation) with predetermined order and applicationinterval during the growing season. When used in a combination with theabove-mentioned products, at a concentration lower than recommended onthe product label, the combined efficacy of the two or more products(one of which is the said composition disclosed herein) is, in certainembodiments, greater than the sum of each individual component's effect.Hence, the effect is enhanced by synergism between these two (or more)products, and the risk for the development of pesticide resistance amongthe plant pathogenic strains is reduced.

The composition can be applied by root dip at transplanting,specifically by treating a fruit or vegetable with the composition bydipping roots of the fruit or vegetable in a suspension of saidcomposition (about 0.25 to about 1.5% and more particularly about 0.5%to about 1.0% by volume) prior to transplanting the fruit or vegetableinto the soil.

Alternatively, the composition can be applied by drip or otherirrigation system. Specifically, the composition can be injected into adrip irrigation system. In a particular embodiment, the composition isapplied in a solution having a concentration of 1×10⁸ CFU/mL at a rateof about 11 to about 4 quarts per acre.

In yet another embodiment, the composition can be added as an in-furrowapplication. Specifically, the composition can be added as an in-furrowspray at planting using nozzles calibrated to deliver a total output of2-6 gallons/acre. Nozzles can be placed in the furrow opener on theplanter so that the pesticide application and seed drop into the furroware simultaneous.

Mixtures of the disclosed compositions with, for example, a solid orliquid adjuvant are prepared as known in the art. For example, mixturescan be prepared by homogeneously mixing and/or grinding the activeingredients with extenders such as solvents, solid carriers and, whereappropriate, surface-active compounds (surfactants). The compositionscan also contain additional ingredients such as stabilizers, viscosityregulators, binders, adjuvants as well as fertilizers or other activeingredients in order to obtain additional desired effects.

Combinations with Plant Growth Promoting Agents

The compositions disclosed herein can be used in combination with othergrowth promoting agents such as synthetic or organic fertilizers (e.g.,di-ammonium phosphate, in either granular or liquid form), compost teas,seaweed extracts, plant growth hormones such as IAA (indole acetic acid)used in a rooting hormone treatment for transplants either alone or incombination with plant growth regulators such as IBA (indole butyricacid) and NAA (naphthalene acetic acid), and growth promoting microbes,such as, for example, methylotrophs, PPFM (Pink Pigmented FacultativeMethylotrphs), Bacillus spp., Pseudomonads, Rhizobia, and Trichoderma.

Seed Treatment/Coating Agents

The compositions disclosed herein can also be used in combination withseed-coating agents. Such seed coating agents include, but are notlimited to, ethylene glycol, polyethylene glycol, chitosan,carboxymethyl chitosan, peat moss, resins and waxes or chemicalfungicides or bactericides with either single site, multisite or unknownmode of action.

The seed treatment methods described herein can be used in connectionwith any species of plant and/or the seeds thereof. In variousembodiments, however, the methods are used in connection with seeds ofplant species that are agronomically important, Ire particular, theseeds can be of corn, peanut, canola/rapeseed, soybean, cucurbits,crucifers, cotton, beets, rice, sorghum, sugar beet, wheat, barley, rye,sunflower, tomato, sugarcane, tobacco, oats, as well as other vegetableand leaf crops. In some embodiments, the seed is corn, soybean, orcotton seed. The seed may be a transgenic seed from which a transgenicplant can grow and incorporates a transgenic event that confers, forexample, tolerance to a particular herbicide or combination ofherbicides, increased disease resistance, enhanced tolerance to stressand/or enhanced yield. Transgenic seeds include, but are not limited to,seeds of corn, soybean and cotton.

Anti-Phytopathogenic Agents

The compositions disclosed herein can also be used in combination withother anti-phytopathogenic agents, such as plant extracts,biopesticides, inorganic crop protectants (such as copper), surfactants(such as rhamnolipids; Gandhi et al., 2007) or natural oils such asparaffin oil and tea tree oil possessing pesticidal properties orchemical fungicides or bactericides with either single site, multisiteor unknown mode of action. As defined herein, an “anti-phytopathogenicagent” is an agent that modulates the growth of a plant pathogen,particularly a pathogen causing soil-borne disease on a plant, oralternatively prevents infection of a plant by a plant pathogen. Plantpathogens include but are not limited to fungi, bacteria, actinomycetesand viruses.

An anti-phytopathogenic agent can be a single-site anti-fungal agentwhich can include but is not limited to benzimidazole, a demethylationinhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole),morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate,quinone outside inhibitor, quinoline, dicarboximide, carboximide,phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon,cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine,phthalimide, benzenoid (xylylalanine). In a more particular embodiment,the antifungal agent is a demethylation inhibitor selected from thegroup consisting of imidazole, piperazine, pyrimidine and triazole(e.g., bitertanol, myclobutanil, penconazole, propiconazole,triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole,hexaconazole, tebuconazole, tetraconazole). In a most particularembodiment, the antifungal agent is myclobutanil. In yet anotherparticular embodiment, the antifungal agent is a quinone outsideinhibitor (e.g., strobilurin). The strobilurin may include but is notlimited to azoxystrobin, kresoxim-methyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether).

In yet a further embodiment, the fungicide is a multi-sitenon-inorganic, chemical fungicide selected from the group consisting ofchloronitrile, quinoxaline, sulphamide, phosphonate, phosphite,dithiocarbamate, chloralkythios, phenylpyridine-amine, andcyano-acetamide oxime.

In yet a further embodiment, the anti-phytopathogenic agent can bestreptomycin, tetracycline, oxytetracycline, copper, or kasugamycin.

Bioremediation

The C. subtsugae genome encodes genes involved in the metabolism of,inter alia, phosphorus, iron and aromatic compounds. See, e.g., Table 6supra. Such genes and their gene products can be used in bioremediationmethods. For instance, genes and sequences related to metal transport,metal accumulation, degradation of organic compounds, and othermetabolite transformation can be engineered into plants with the purposeof applying the transformed plant to bioremediation of soils, sediment,water, and other polluted substrates. Protocols for the transformationof Indian mustard (Brassica juncea), sunflower (Helianthus annus),tomato and yellow poplar (Liriodendron tulipifera) are known. See, e.g.,Eapen and D'Souza (2005); Mello-Farias and Chavez (2008).

Plants can be transformed with Cytochrome P450-encoding genes toincrease their resistance to particular pollutants, both organic andinorganic. Transformation with nucleic acids encoding enzymes involvedin gluthatione conjugation (for example, glutathione S-transferases) canincrease rates of xenobiotic detoxification. Plants expressing bacterialnitroreductases can be used for the detoxification of nitrate organiccompounds, such as explosives.

Uses of transgenic plants for phytoremediation applications has beendescribed, for example, by Abhilash et al. (2009); Van Aken et al.(2010); Doty (2008) and Macek et al. (2008).

Table 2 below denotes additional information of the present disclosureon Chromobacterium substague proteins.

TABLE 2 Apparent MW Protein (SDS- Amino Acid name Function PAGE)Nucleotide sequences Sequences Homology Scott1~20 kDa >fig|6666666.22288.peg.2223 MSLTTDFLEN Extracellular,[MBI-203 sp.][hypothetical PQAFMRSQAI Secreted. protein] LIPAQVPPGNatgtctttgactaccgattttctggagaacc GKYQFAAQGAcgcaggctttcatgcgttctcaggcgatatt HAAVLQSTAAgattccggcgcaggttccgcccggcaacggc SPNIPGFYAHaaataccagttcgcggcgcagggcgcgcacg PVANNINLFVcggcggtgctgcagtccaccgccgccagccc LPTQQPARYYgaacatccccggtttttacgcgcatccggtg MFTDGMNGCQgccaacaacatcaatctgttcgtgctgccca FLAYGPDRQHcccagcagccggcaaggtattacatgttcac ITVEHNNFIGcgacggcatgaacggctgccagtttctggcc DPTRYAARLAtacgggccggacaggcagcacatcaccgtcg EVVALKPAYLagcacaacaacttcatcggcgacccgacgcg LHISPSGVNNctacgcggcgcggctggcggaggtggtggcg IPAGQYNSQQctgaagccggcctatctgctgcatatcagcc GVNIVGEYGQcgtcgggggtcaataacatcccggccggcca ANGWRFWVRDatacaacagccagcagggggtgaacatcgtc RVDQNQGTVYggcgaatacggccaggccaacggctggcgct GPL tctgggtgcgggacagggtggaccagaacca(SEQ ID NO: 1) gggcacggtgtacgggccgctgtaa (SEQ ID NO: 4) Scott2 Protease~20 kDa >fig|6666666.22288.peg.3563 MDKRLPAVAA Protease BLASTp[MBI-203 sp.][probable protease ALLLAASAAH C. violaceum precursor]AGDLQVSLGQ M35 peptidase atggacaagagattgccagccgtggccgcgg PVVSAGQDVDdomain, Lysine- ctttgctgttggcggcttccgccgctcacgc VALTYRNTGKspecific metallo- cggcgacctgcaggtcagcctggggcagccg ETLHVYRWFVendopeptidase, gtggtcagcgcgggtcaggacgttgacgtcg PGKELQEQFL Zn bindingctttgacttaccgcaataccggcaaggagac AVNVNGKPAE domain.cttgcacgtgtaccgctggttcgtgcccggc YLGPRYKRVVaaggaactgcaggagcagtttctggcggtga PSLRDTVALAatgtgaacggcaagccggccgagtacctggg PGATLNAKVRcccgcgctacaagcgcgtggtgccgtcgctg VSEYYDLSKPcgcgacaccgtggcgctggcgcccggcgcca GQLSVRFESScgctgaacgccaaggtgagggtgtccgagta SNKVLNRSLPttacgacctgtccaagccgggccagctgagc AGVNAKQAAAgtccgttttgaaagcagcagcaacaaggtgc PQADEAISSNtcaaccgcagcctgccggccggcgtcaatgc VVGAYSAGSVcaagcaggcggccgcgccgcaggccgacgag SPLLTKSKAAgccatttcctccaatgtggtgggcgcctaca KQEWQVLSRSgcgccggcagcgtcagccccttgctgaccaa AVSGVSYAGNgtccaaggcggccaagcaggagtggcaggtg CSVSQQSQSRctcagccgcagcgcggtcagcggcgtcagct DGVLAASAMAacgccggcaattgctcggtcagccagcagtc SETAAYLAGTgcaatcgcgcgacggcgtgctggccgccagc PSGTPRFTTWgccatggccagcgaaacggcggcctacctgg FGKYSQANWTccggcacgccgtccggcacgccgcgcttcac TAKSHYVNIKcacttggttcggcaagtacagccaggccaac DALDSKPIKLtggaccaccgccaagtcgcattacgtcaaca DCSCTDGGTYtcaaggatgcgctggacagcaagccgatcaa AYVYPGQPYTgctggattgcagctgcaccgacggcggcact VYLCGAFWTAtatgcctacgtctatccggggcagccgtaca PTKGTDSKGGccgtctatctgtgcggcganctggaccgcgc TLVHELSHFTcgaccaagggcaccgactccaagggcggcac VVAGTQDHVYcctggtgcatgagttgtcgcacttcaccgtg GQAGAKSLAKgtggcgggcacccaggaccatgtctatggcc SNPAQALDNAaggccggcgccaagagcctggccaagagcaa DNHEYFAENTcccggcccaggccttggacaatgccgacaac PAQQ catgaatacttcgccgagaacaccccggcgc(SEQ ID NO: 2) agcagtaa (SEQ ID NO: 5) Scott3 Metallo-~34 kDa >fig|6666666.22288.peg.175  MRKQQLMLRG Class IV protease[MBI-203 sp.][Vibriolysin, LVLSALAVFS metalloprotease;extracellular zinc protease SAALAAERID vibriolysin, (EC 3.4.24.25) @Pseudolysin, LEKLGKIQAN pseudolysin extracellular zinc GAVAFTGVNQExtracellular, protease (EC 3.4.24.26)] ADLKPLRSTQ Zinc proteaseatgagaaaacagcaattgatgttgcgtggtt FATGKVVTRFtggtcctgtccgccctggctgtgttcagctc QQYYQGVPVWggcggcgctggcggccgagcgtatcgacctg GEAVVEEKQAgaaaagctgggcaagatccaggccaacggcg GAVAKTSGKLcggtggcgttcaccggcgtgaaccaggctga SGQYIAGIQStctgaagcccctgcgcagcacccaattcgcc DLASAKPTLSaccggcaaagtggtgacccgcttccagcagt SAQALSQAKSactaccagggcgtgccggtatggggcgaagc LKANGNPTYNcgtggtcgaggaaaaacaggccggcgccgtg EKADLVVRLNgccaagaccagcggcaaactatccggccaat ERNTAQLVYLacatcgccggcatccagtccgacctggcttc VSFVVDGKEPcgccaagccgacgttgagcagcgcccaggcg SRPHLIIDANttgagccaggccaaatcgctgaaggccaacg NGQVLKQWEGgcaatcccacctacaacgagaaagccgacct LNHAEANGPGagtggtgcgcctgaacgagcgcaacaccgcc GNAKTGKYVYcagctggtctacctagtgtccttcgtggtcg GTDYGPLIVTacggcaaggagcccagccgcccgcacctgat SDCKMDSGNVcatcgacgccaacaacggccaggtgctgaag ATVNLNGGTScagtgggaaggcctgaaccacgccgaagcca GTTPYKFACPacggccccggcggcaacgccaagaccggcaa TNTYKAINGAgtatgtctacggcaccgactacggtccgctg YSPLNDAHYFatcgtcaccagcgattgcaagatggatagcg GNVVFNLYKDgcaacgtcgccaccgtcaacctcaacggcgg WFNLKPINQKcaccagcggcaccaccccgtacaagttcgcc LLMKVHYSRNtgcccgaccaacacctacaaagcgatcaacg YENAFWDGTAgcgcttactcgccgctgaacgacgcgcacta MTFGDGASTFcttcggcaacgtggtgttcaacctgtacaag YPLVSLDVSAgactggttcaacctgaagccgatcaaccaga HEVSHGFTEQagctgctgatgaaggtgcactacagccgcaa NSGLVYDGQSctacgaaaacgcgttctgggacggcaccgcg GGINEAFSDMatgaccttcggcgacggcgccagcaccttct AGEAAEYYMKacccgctggtgtcgctggacgtgtccgcgca GKNDFLVGAEtgaagtcagccacggcttcaccgagcagaac IFKKTGALRYtccggcctggtctacgacggccagtccggcg FADPTKDGQSgcatcaacgaggcattctccgacatggccgg IGNAKDYYDGcgaagccgccgagtactacatgaagggcaag LDVHYSSGVYaacgacttcctggtgggcgcggaaatcttca NKAFYLIATSagaagaccggcgcgctgcgctacttcgccga PNWNTRKAFEtccgaccaaggacggccaatcgatcggcaac VFVDANRLYWgccaaggactactacgacggcctggacgtgc TANATYNSAAactattccagcggcgtgtacaacaaggcctt CGVVKAADARttacctgatcgccaccagcccgaactggaac GYNSADVTKAacccgcaaggcgtttgaagtgttcgtcgacg FTAVGVTCKccaaccggctgtactggaccgccaacgccac (SEQ ID NO: 3)ctacaacagcgccgcttgcggcgtggtcaag gcggccgacgcccgcggctacaacagcgccgacgtcaccaaggccttcaccgcagtcggcgt gacttgcaaataa (SEQ ID NO: 6)

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLES Example 1: Cell Growth and DNA Extraction

Chromobacterium subtsugae PRAA-1 was grown in 200 ml LB broth in 1 Lflasks at 26° C. with rotation at 150 rpm for 24-48 hours. Biomass washarvested from the culture by centrifugation.

Genomic DNA was extracted using the MoBio Power Microbial Maxi-DNAExtraction Kit (MoBio Cat No. 122223-25). DNA was eluted in 1.5 ml ofelution buffer (included in kit). To assess DNA quality and quantity, a10 uL aliquot was loaded into a 1.5% agarose gel and electrophoresis wasconducted for 30 minutes at 100 V. DNA was visualized with a UVtransilluminator using EZ-Vision loading dye. Over 100 ug of DNA wererecovered.

Example 2: DNA Sequence Determination and Assembly

DNA sequences were determined using a HiSeq 2000 (Illumina, San Diego,Calif.), with sequence reads of 100 bp, pair ended, aiming for a minimumcoverage of 40×. Final data consisted of two sets of paired-end samplesin FASTQ format, providing approximately 200× coverage of the genome.

The four FASTAQ files were used for assembly. FASTAQ sequences weresubjected to quality control using FASTQC, and the average distancebetween pairs was calculated by comparing the first 10,000 pairs fromboth sets with the initial assembled contigs using BWA. Li & Durbin(2009) Bioinformatics 25(14):1754-1760. TrimGalore (BabrahamBioinformatics, Cambridge, UK) was then used to generate twohigh-quality paired-end sets and four single-read files for thosesequences whose partner read was below the quality threshold of at least50 nucleotides after clipping on Q2.

Sequence reads were assembled using Ray assembler v2.0.0. Boisvert etal. (2010) J Comput Biol. 17(11):1519-1533. A titration of kmer sizeswas performed with a kmer range of 19-63; resulting in successfulassemblies at 19, 21, 31, 41, 47, 49 and 63. Further scaffolding wasperformed using SSPACE v1.1 using all available reads on the scaffoldsproduced by the Ray analysis. Boetzer et al. (2011) Bioinformatics27(4):578-579. Gaps were connected using GapFiller, with a maximumiteration of twenty steps. Boetzer & Pirovano (2012) Genome Biol.13(6):R56. The resulting scaffolds were mapped against the referencegenome of Chromobacterium violaceum ATCC 12742, using CONTIGuator withan e value of 1e-10. Galardini et al. (2011) Source Code Biol. Med.6:11.

To confirm contig and scaffold orders, the alignments were inspectedmanually using ACT. Carver et al. (2008) Bioinformatics24(23):2672-2676. The original dataset was mapped back onto theChromobacterium subtsugae sequence using BWA (Li & Durbin, supra) with aseed length of 19.

This process yielded a high quality genome of 4,690,330 bases with atotal of 145,992 bases in contigs not matching the reference genome(Chromobacterium violaceum) and 4,264 undefined nucleotides (N's) in 42gaps. Subsequent filling of the gaps in pseudocontigs closed 8 of the 42gaps and extended the pseudocontigs to 4,704,820 bases where most gapsare single ‘N’ positions with only 2 gaps remaining in positions2,153,178-2,153,283 (105 bases) and 2,474,439-2,474,486 (47 bases).

Example 3: Screening of Proteins for Cabbage Loopers InsecticideActivity

Proteins Scott 1-3 (Seq ID Nos: 4-6, respectively) were fractionated bystandard FPLC procedure.

Diet plates were stored in a covered container in the bioassayrefrigerator (4° C.). All efficacy tests were conducted with a minimumof six dilutions and two dilution replicates (wells) per plate and aminimum of 40 wells total for each dilution. Deionized water was used asnegative control. All protein candidates and the standard were seriallydiluted to a minimum of six dilutions. (i.e. 16%, 8%, 4%, 2%, 1% and0.05%). Starting with the lowest dilution, 100 μL of each dilutiontreatment was pipetted into each well. The plate was moved into a fumehood with a small room fan. The fan was turned on and pointed at theplate in an angle that the liquid in the wells are affected by theairflow. Wells were sufficiently dry so that a neonate larva can beplaced into each well without drowning. Only first through early secondinstar Cabbage Loopers were used for the 96-well plates. Using a finepaintbrush, single small larva was moved from its rearing area into awell. Infestation was continued until all wells have larvae in them. Asmall hole was made into the lid over each well for ventilation, using adental pick or other small pointed tool. The plate was placed in acontrolled-temperature chamber at 26° C., and mortality was scored on 3to 4 days after addition of insects. Insect mortality was determined byexamining the larvae in each well. Average mortality for each dilutionwas then determined. Tables 3-6 show the mortality of cabbage loopersachieved by the amount of Scott 1-3 (SEQ ID Nos:4-6).

TABLE 3 (Scott1; SEQ ID No: 1 or 4) % mortality Corrected % mortalitySample description Day 3 Day 4 Day 3 Day 4 Captured scott1 15.00 25.0012.821 23.077 fraction, 0.22 mg/mL Captured scott1 5.00 5.00 2.564 2.564fraction, 0.11 mg/mL Captured scott1 5.00 5.00 2.564 2.564 fraction,0.06 mg/mL Captured scott1 5.00 5.00 2.564 2.564 fraction, 0.03 mg/mLNegative control 2.50 2.50 (water) Captured scott1 9.17 8.33 9.167 8.333fraction, 0.224 mg/mL Captured scott1 16.67 20.83 16.667 20.833fraction, 0.448 mg/mL Captured scott1 41.25 65.42 41.250 65.417fraction, 0.896 mg/mL Negative control 0.00 0.00 (water) Partiallypurified 31.67 53.70 28.696 51.691 scott1, 2.9 mg/mL Partially purified46.67 94.44 44.348 94.203 scott1, 5.7 mg/mL Partially purified 48.3388.89 46.087 88.406 scott1, 11.5 mg/mL Negative control 4.17 4.17(water)

TABLE 4 (scott2; SEQ ID No: 2 or 5) % mortality Corrected % mortalitySample description Day 3 Day 4 Day 3 Day 4 Partially purified 20k 33.3333.33 30.43 30.43 protease 0.5 mg/mL Partially purified 20k 55.56 72.2253.62 71.01 protease 0.5 mg/mL w/ 10 mM Zn and Ca Negative control 4.174.17 (water) Partially purified 20 kDa 71.33 83.00 71.33 82.11 protease0.5 mg/mL w/ 10 mM Zn and Ca Negative control 0.00 5.00 (water)Partially purified 20 kDa 22.22 22.22 21.13 20.00 protease 0.5 mg/mLNegative control 1.39 2.78 (water) Partially purified 20 kDa 22.22 22.2219.71 17.65 protease 0.5 mg/mL Negative control 3.13 5.56

TABLE 5 (scott3, SEQ ID No: 3 or 6) % mortality Corrected % mortalitySample description Day 3 Day 4 Day 3 Day 4 Partially purified 35 kDa7.41 7.41 3.38 3.38 protease, 0.25 mg/mL Partially purified 35 kDa 18.5224.07 14.98 20.77 protease, 0.25 mg/mL w/ 10 mM Zn and Ca Negativecontrol 4.17 4.17 (water) Partially purified 35 kDa 0.00 0.00 0.00 −5.26protease, 0.125 mg/mL Partially purified 35 kDa 4.00 6.67 4.00 1.75protease, 0.125 mg/mL w/ 10 mM Zn and Ca Negative control 0.00 5.00(water) Partially purified 35 kDa 74.07 74.07 73.71 73.33 protease 0.5mg/mL Negative control 1.39 2.78 (water) Partially purified 35 kDa 51.8557.41 50.30 54.90 protease 0.5 mg/mL Negative control 3.13 5.56 (water)

Example 4: Transformation of Tomato (Solunum lycopersicum) withAgrobacterium

The following procedure is adapted from Sharma, M. K. et al. 2009. “Asimple and efficient Agrobacterium-mediated procedure for transformationof tomato.” Journal of Biosciences 34:423-433.

Media and Solutions

The composition of various media is described in Table 6. Mediacomponents, except agar, are combined according to Table 6 and adjustedto pH 5.8 using 1N KOH, before adding plant-tissue culture grade agar.Stock solutions of BAP (6-benzylmaino purine) and zeatin are prepared indimethyl sulphoxide (DMSO). Antibiotic stock solutions are prepared indeionized water and filter-sterilized. Agrobacterium strain AGL1 isgrown on YEM agar or broth containing 100 mg/l rifampicin and 50 mg/Ikanamycin.

Preparation of Agrobacterium

Agrobacterium tumefaciens, transformed with the gene or genes ofinterest, (e.g., any of the genes disclosed in SEQ ID NOs:1-6) is grownin YEM medium with rifampicin and kanamycin, in shaking culture for 72 hat 28° C. and 200 rpm. Cells are pelleted by centrifugation, washed andre-suspended in WS medium. Bacterial density is determined by measuringOD₆₀₀ and the final cell concentration is adjusted to ˜10⁸ cells/ml bydiluting with WS medium.

Plant Transformation

Middle pieces (0.7×1.0 cm) from 10-day cotyledons are collected byexcising at the tip and base. The sections are pre-cultured for 48 hoursat 28° C. on Ml medium, with the adaxias surface in direct contact withthe medium.

Healthy explants are selected and incubated in Agrobacterium suspensionfor 30 minutes, with inversion every 10 minutes. Explants are blotted onsterile tissue paper and returned to Ml agar (50-80 explants per plate)for an additional 72 hours. The explants are then washed 4-5 times in WSmedium, blotted on sterile tissue paper and transferred to SM containing1 mg/L trans-zeatin for regeneration (20-25 explants per regenerationplate).

Regeneration plates are incubated at 28° C. under a 16/8 light/darkcycle. Regeneration is evidenced by development of a callus. Regeneratedexplants are selected and transferred to fresh SM medium every 15 days.

Regenerated shoots can be excised from the callus and transferred to RMmedium.

Plantlets that are at least 2 inches in height and have strong roots areselected for transfer to pots. Planting substrate consists of pottingsoil mixed 1:1 with 1:1:1 vermiculite:perlite:sphagnum.

TABLE 6 M1 M2 WS SM RM MS Salts 0.5x 1x 1x 1x 1x (Murashige and Skoog,1962) Gamborg's B5 0.5x 1x 1x 1x 1x vitamins Sucrose (g/L) 15 30 30 3030 Agar (% w/v) 0.8 0.8 0 0.8 0.8 BAP (mg/L) 0 2 0 0 0 Kanamycin (mg/L)0 0 0 100 100 Cefotaxime (mg/L) 0 0 0 500 500

Example 5: Creation of Transgenic Soybean Plants Comprising anInsecticidal Gene from Chromobacterium substugae

Mature glycine max seeds are surface sterilized with chlorine gas insidea bell jar under a fume hood. Seeds are kept in 100×20 mm Petri disheswith chlorine gas produced by pouring 100 ml of 4% sodium hypochloriteinto a beaker and adding 5 ml of 12N hydrochloric acid. Aftersterilization, seeds are placed on germination medium (GM; MS basalsalts with vitamins, 3% sucrose, 0.8% plant agar, and 1 mg/L BAP,optimized from regeneration experiment, pH 5.8). Murashige and Skoog,1962. Seeds are germinated under fluorescent light or darkness at 24±1°C. for 5-7 days to compare transformation frequency.

The method described here is a modification of that described by Zhanget al. (1999) Plant Cell, Tissue and Organ Culture 56:37-46. Twocotyledonary explants are obtained by cutting a horizontal slice throughthe hypocotyl with a No. 11 surgical blade. The hypocotyl issubsequently removed and ten scratches are made at the surface ofcotyledonary node regions. Explants are immersed for 30 min in asuspension of A. tumefaciens which has been engineered to comprise thegene of interest, e.g., a gene that encodes an insecticidal protein, ora protein that is involved in the synthesis of an insecticidal compound.See Tables 2 above for listings of exemplary genes of interest.Following immersion, ten explants are randomly placed on sterile filterpaper placed on solid co-cultivation medium (CM; Gamborg's B5 basalsalts with vitamins, 3% sucrose, 20 mM MES, 3.3 mM L-cysteine, 1 mMdithiothreitol, 0.1 mM acetosyringone, 0.8% plant agar, pH 5.4) (Gamborget al., 1968) in 100×20 mm Petri dishes, and incubated at 24±1° C. for 5days under dark conditions.

After 5 days of co-cultivation, explants are briefly washed in liquidshoot induction medium (SIM; Gamborg's B5 basal salts with vitamins, 3%sucrose, 3 mM MES, 1.67 mg/L BAP, 250 mg/L cefotaxime, pH 5.7) to removeexcess A. tumefaciens on explants. Explants are then transferred tosolidified SIM without PPT to stimulate shoot induction for the first 14days, after which the explants are sub-cultured on fresh SIM containing5 mg/L PPT for selection of transformed shoots. Organogenic shoots fromthe explants are trimmed and then transferred to shoot elongation medium(SEM; MS basal salts with vitamins, 3% sucrose, 3 mM MES, 0.5 mg/Lgiberellic acid, 50 mg/L asparagine, 1 mg/L zeatin, 0.1 mg/Lindole-3-acetic acid, 250 mg/L cefotaxime, 50 mg/L vancomycin, 0.8%plant agar, 5 mg/L PPT, pH 5.7). Explants are transferred to new SEMmedium every 14 days, and surviving shoots are planted on root inductionmedium (RIM; MS basal salts with vitamins, 3% sucrose, 1 mg/Lnaphthalene acetic acid, 0.8% plant agar, pH 5.7) and grown until rootsdevelop. After acclimation, the transgenic plants are transplanted topotting soil and maintained in a greenhouse. Selection is carried out byPCR. See also Lee, et al. (2011) J. of Korean Soc. Appl. Biol. Chem. 54:37-45.

Example 6: Screening of Proteins for Cabbage Loopers (Trichoplusia ni)(CL), Lygus (Lygus hesperus), Beet Armyworms (Spodoptera exigua)(BAW),and Diamondback Moth (Plutella xylostella) (DBM) Insecticidal Activity

Proteins were partially purified by strong cation exchange andhydrophobic interaction chromatography. Protein concentration wasestimated using the Invitrogen Quant-iT or Qubit assay calibrated withBSA. Unless otherwise noted, scott1 (SEQ ID NO:1 or 4) was submitted forbioassay at 5 mg/mL, scott2 (SEQ ID NO:2 or 5) at 1 mg/mL, and scott3(SEQ ID NO:3 or 6) at 1 mg/mL total protein amounts. Proteins werebuffered at pH 6 in 20 mM MES or pH 7.5 with 20 mM Tris. Scott2 wasspiked with up to 5 mM ZnCl₂ and CaCl₂.

Activity against Cabbage Loopers (repeated as compared to example 3),Beet Armyworm and Diamondback Moth was tested on Diet Overlay Bioassays.The appropriate artificial insect diet was dispensed into each well of astandard 96 well plate and allowed to dry. Once the diet solidified, 100uL of the treatment was pipetted into the appropriate number of wellsand allowed to dry. A single 1st instar larva was delivered into eachwell of a 96 well plate. Mortality was scored at 4 days after treatment.

Activity against lygus was tested on an Artificial Diet Bioassay asfollows: Diet packets were prepared by combining the appropriate amountof lygus artificial diet and stock treatment solution. The mixtures werevortexed and distributed evenly amongst the diet packets. Nymphs, 10-12lygus 2nd or 3rd instar, were placed into a petri dish, covered with amesh lid and sealed with Parafilm. Mortality was scored at 4 days afterexposure to the treated diet.

Efficacy is expressed as percentage mortality at 4 days post treatment.Results (in duplicates) in % mortality are shown in Table 7. Scott1 wasprepared at 4.9 mg/mL for the first lygus assay. Scott3 was prepared at0.42 mg/mL for the first lygus assay and at 0.82 mg/mL for the firstcabbage looper assay.

TABLE 7 CL % Mortality Lygus BAW DBM Scott1 90.91 100 21.98 12.5 8.33 095.83 77.08 Scott2 100 92.86 22.88 19.3 25 33.33 58.33 95.83 Scott363.64 100 7.54 15.21 29.17 33.33 95.83 70.83

The inventions described and claimed herein are not to be limited inscope by the specific aspects herein disclosed, since these aspects areintended to be illustrative. Any equivalent aspects are intended to bewithin the scope of the disclosure. Indeed, various modifications of themethods and compositions shown and described herein will be apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims. In the case of conflict, the present disclosure includingdefinitions will control.

What is claimed is:
 1. A a recombinant vector comprising a heterologouspromoter operably linked to a nucleotide sequence encoding a polypeptidewith 100% identity to SEO ID NO:
 1. 2. A plant or plant parts, orprogeny or seed thereof, comprising the recombinant vector of claim 1.3. The plant or plant parts of claim 2, wherein said plant part isselected from the group consisting of pollen, ovule, flower, shoot,root, stalk, silk, tassel, ear, and leaf tissue.
 4. A cell comprisingthe recombinant vector of claim
 1. 5. The cell of claim 4, wherein saidcell is a bacterial, mammalian, or fungal cell.
 6. A method of producingan insect resistant plant comprising the step of transforming therecombinant vector of claim 1 into a plant cell.
 7. Anti-counterfeitmilled seed having, as an indication of origin, a plant cell comprisingthe recombinant vector of claim 1.